U.S. patent application number 16/382692 was filed with the patent office on 2019-10-24 for novel phosphorous (v)-based reagents, processes for the preparation thereof, and their use in making stereo-defined organophosho.
The applicant listed for this patent is Bristol-Myers Squibb Company, The Scripps Research Institute. Invention is credited to Phil BARAN, Justine deGRUYTER, Martin D. EASTGATE, William R. EWING, Kyle KNOUSE, Ivar M. McDONALD, Richard E. OLSON, Michael Anthony SCHMIDT, Bin ZHENG.
Application Number | 20190322694 16/382692 |
Document ID | / |
Family ID | 66323965 |
Filed Date | 2019-10-24 |
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United States Patent
Application |
20190322694 |
Kind Code |
A1 |
SCHMIDT; Michael Anthony ;
et al. |
October 24, 2019 |
NOVEL PHOSPHOROUS (V)-BASED REAGENTS, PROCESSES FOR THE PREPARATION
THEREOF, AND THEIR USE IN MAKING STEREO-DEFINED ORGANOPHOSHOROUS
(V) COMPOUNDS
Abstract
The present invention relates to novel phosphorous (V) (P(V))
reagents, methods for preparing thereof, and methods for preparing
organophosphorous (V) compounds by using the novel reagents.
Inventors: |
SCHMIDT; Michael Anthony;
(Cranbury, NJ) ; ZHENG; Bin; (Kendall Park,
NJ) ; KNOUSE; Kyle; (New Oxford, PA) ;
deGRUYTER; Justine; (College Station, TX) ; EASTGATE;
Martin D.; (Titusville, NJ) ; BARAN; Phil;
(San Diego, CA) ; EWING; William R.; (Yardley,
PA) ; OLSON; Richard E.; (Cambridge, MA) ;
McDONALD; Ivar M.; (Woodstock, CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bristol-Myers Squibb Company
The Scripps Research Institute |
Princeton
La Jolla |
NJ
CA |
US
US |
|
|
Family ID: |
66323965 |
Appl. No.: |
16/382692 |
Filed: |
April 12, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62729314 |
Sep 10, 2018 |
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62697896 |
Jul 13, 2018 |
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62668098 |
May 7, 2018 |
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62657551 |
Apr 13, 2018 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07F 9/65742 20130101;
C07H 1/02 20130101; C07H 19/10 20130101; C07H 21/00 20130101; C07H
19/11 20130101; C07H 19/213 20130101; C07F 9/65744 20130101 |
International
Class: |
C07H 21/00 20060101
C07H021/00 |
Claims
1. A compound of the formula (I): ##STR00513## wherein (a) R.sup.1,
R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are independently
hydrogen, CD.sub.3 or CF.sub.3, linear or branched C.sub.1-C.sub.20
alkyl, optionally substituted with one or more, the same or
different, R.sup.a groups; linear or branched C.sub.2-C.sub.12
alkenyl; optionally substituted with one or more, the same or
different, R.sup.a groups; linear or branched C.sub.2-C.sub.12
alkynyl, optionally substituted with one or more, the same or
different, R.sup.a groups; aryl, optionally substituted with one or
more, the same or different, R.sup.a groups; heteroaryl, optionally
substituted with one or more, the same or different, R.sup.a
groups; heterocyclyl, optionally substituted with one or more, the
same or different, R.sup.a groups; or C.sub.3-C.sub.8 cycloalkyl,
optionally substituted with one or more, the same or different,
R.sup.a groups; or (b) any two of R.sup.1, R.sup.2, R.sup.3,
R.sup.4, R.sup.5, and R.sup.6 together with the carbons to which
they are attached form a C.sub.4-C.sub.8 cycloalkyl group,
optionally substituted with one or more, the same or different,
R.sup.a groups, while the remaining R.sup.1, R.sup.2, R.sup.3,
R.sup.4, R.sup.5, and R.sup.6 are as defined in (a); R.sup.a is
hydrogen; deuterium, CD.sub.3, C.sub.1-C.sub.6 alkyl, OH, halogen,
CN, CF.sub.3, O--C.sub.1-C.sub.6 alkyl, O-aryl, O-heteroaryl,
O--C.sub.3-C.sub.8 cycloalkyl, O-heterocyclyl, --NR.sup.bR.sup.b,
--COOR.sup.b or --CONR.sup.bR.sup.b; R.sup.b is independently at
each occurrence, the same or different, hydrogen, linear or
branched C.sub.1-C.sub.12 alkyl, aryl, heteroaryl, heterocyclyl or
C.sub.3-C.sub.8 cycloalkyl; X.sup.1 and X.sup.2 are independently
O, S or NR.sup.c; wherein R.sup.c is hydrogen or C.sub.1-C.sub.4
alkyl, Y is O, S or NR.sup.c; m is 0, 1 or 2, and LG is a leaving
group.
2. The compound according to claim 1, having any one of formulae
(II)-(IIc): ##STR00514## wherein (a) R.sup.1, R.sup.3, R.sup.5 and
R.sup.6 are independently hydrogen, CD.sub.3 or CF.sub.3, linear or
branched C.sub.1-C.sub.20 alkyl, optionally substituted with one or
more, the same or different, R.sup.a groups; linear or branched
C.sub.2-C.sub.12 alkenyl; optionally substituted with one or more,
the same or different, R.sup.a groups; linear or branched
C.sub.2-C.sub.12 alkynyl, optionally substituted with one or more,
the same or different, R.sup.a groups; aryl, optionally substituted
with one or more, the same or different, R.sup.a groups;
heteroaryl, optionally substituted with one or more, the same or
different, R.sup.a groups; heterocyclyl, optionally substituted
with one or more, the same or different, R.sup.a groups; or
C.sub.3-C.sub.8 cycloalkyl, optionally substituted with one or
more, the same or different, R.sup.a groups; or (b) any two of
R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 together
with the carbons to which they are attached form a C.sub.4-C.sub.8
cycloalkyl group, optionally substituted with one or more, the same
or different, R.sup.a groups, while the remaining R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are as defined in (a);
R.sup.a is hydrogen; deuterium, CD.sub.3, C.sub.1-C.sub.6 alkyl,
OH, halogen, CN, CF.sub.3, O--C.sub.1-C.sub.6 alkyl, O-aryl,
O-heteroaryl, O--C.sub.3-C.sub.8 cycloalkyl, O-heterocyclyl,
--NR.sup.bR.sup.b, --COOR.sup.b or --CONR.sup.bR.sup.b; R.sup.b is
independently at each occurrence, the same or different, hydrogen,
linear or branched C.sub.1-C.sub.12 alkyl, aryl, heteroaryl,
heterocyclyl or C.sub.3-C.sub.8 cycloalkyl; Y is O, S or NR.sup.c,
where R.sup.c is hydrogen or C.sub.1-C.sub.4 alkyl; m is 0, 1 or 2,
and LG is a leaving group; provided that either the carbon bearing
the R.sup.1 and R.sup.2 groups, the carbon bearing the R.sup.3 or
and R.sup.4 groups, or both, is chiral; wherein the compound of
formula (I) is at least 90% stereochemically pure.
3. (canceled)
4. (canceled)
5. The compound of claim 1 of the formula ##STR00515## wherein
R.sup.2 and R.sup.4 are independently hydrogen, CD.sub.3 or
CF.sub.3, linear or branched C.sub.1-C.sub.20 alkyl, optionally
substituted with one or more, the same or different, R.sup.a
groups; linear or branched C.sub.2-C.sub.12 alkenyl; optionally
substituted with one or more, the same or different, R.sup.a
groups; linear or branched C.sub.2-C.sub.12 alkynyl, optionally
substituted with one or more, the same or different, R.sup.a
groups; aryl, optionally substituted with one or more, the same or
different, R.sup.a groups; heteroaryl, optionally substituted with
one or more, the same or different, R.sup.a groups; heterocyclyl,
optionally substituted with one or more, the same or different,
R.sup.a groups; or C.sub.3-C.sub.8 cycloalkyl, optionally
substituted with one or more, the same or different, R.sup.a
groups; R.sup.a is hydrogen, deuterium, CD.sub.3, C.sub.1-C.sub.6
alkyl, OH, halogen, CN, CF.sub.3, O--C.sub.1-C.sub.6 alkyl, O-aryl,
O-heteroaryl, O--C.sub.3-C.sub.8 cycloalkyl, O-heterocyclyl,
--NR.sup.bR.sup.b, --COOR.sup.b or --CONR.sup.bR.sup.b; R.sup.b is
independently at each occurrence, the same or different, hydrogen,
linear or branched C.sub.1-C.sub.12 alkyl, aryl, heteroaryl,
heterocyclyl or C.sub.3-C.sub.8 cycloalkyl; R.sup.7 is hydrogen;
CD.sub.3, OH, halogen, CN, CF.sub.3, linear or branched
C.sub.1-C.sub.6 alkyl; linear or branched C.sub.2-C.sub.6 alkenyl,
or linear or branched C.sub.2-C.sub.6 alkynyl; X.sup.1 and X.sup.2
are independently O, S or NR.sup.c, wherein R.sup.c is hydrogen or
C.sub.1-C.sub.4 alkyl; Y is O, S or NR.sup.c; and LG is a leaving
group
6. (canceled)
7. (canceled)
8. The compound according to claim 5, having the formula
##STR00516## wherein R.sup.2 and R.sup.4 are independently
hydrogen, CD.sub.3, CF.sub.3, linear or branched C.sub.1-C.sub.20
alkyl, optionally substituted with one or more, the same or
different, R.sup.a groups; linear or branched C.sub.2-C.sub.12
alkenyl; optionally substituted with one or more, the same or
different, R.sup.a groups; linear or branched C.sub.2-C.sub.12
alkynyl, optionally substituted with one or more, the same or
different, R.sup.a groups; aryl, optionally substituted with one or
more, the same or different, R.sup.a groups; heteroaryl, optionally
substituted with one or more, the same or different, R.sup.a
groups; heterocyclyl, optionally substituted with one or more, the
same or different, R.sup.a groups; or C.sub.3-C.sub.8 cycloalkyl,
optionally substituted with one or more, the same or different,
R.sup.a groups; R.sup.a is hydrogen, deuterium, CD.sub.3,
C.sub.1-C.sub.6 alkyl, OH, halogen, CN, CF.sub.3,
O--C.sub.1-C.sub.6 alkyl, O-aryl, O-heteroaryl, O--C.sub.3-C.sub.8
cycloalkyl, O-heterocyclyl, --NR.sup.bR.sup.b, --COOR.sup.b or
--CONR.sup.bR.sup.b; R.sup.b is independently at each occurrence,
the same or different, hydrogen, linear or branched
C.sub.1-C.sub.12 alkyl, aryl, heteroaryl, heterocyclyl or
C.sub.3-C.sub.8 cycloalkyl; provided that either the carbon bearing
the R.sup.1 or R.sup.2 groups, the carbon bearing the R.sup.3 or
R.sup.4 groups, or both, is chiral; X.sup.1 and X.sup.2 are
independently O, S or NR.sup.c; Y is O, S or NR.sup.c; R.sup.c is
hydrogen or C.sub.1-C.sub.4 alkyl; and LG is a leaving group.
9. (canceled)
10. (canceled)
11. The compound according to claim 2, selected from the group
consisting of ##STR00517##
12. (canceled)
13. The compound according to claim 8, selected from the group
consisting of ##STR00518##
14. A stereochemically pure compound of formula (IV): ##STR00519##
wherein (a) R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and
R.sup.6 are independently hydrogen, CD.sub.3, CF.sub.3, linear or
branched C.sub.1-C.sub.20 alkyl, optionally substituted with one or
more, the same or different, R.sup.a groups; linear or branched
C.sub.2-C.sub.12 alkenyl; optionally substituted with one or more,
the same or different, R.sup.a groups; linear or branched
C.sub.2-C.sub.12 alkynyl, optionally substituted with one or more,
the same or different, R.sup.a groups; aryl, optionally substituted
with one or more, the same or different, R.sup.a groups;
heteroaryl, optionally substituted with one or more, the same or
different, R.sup.a groups; heterocyclyl, optionally substituted
with one or more, the same or different, R.sup.a groups; or
C.sub.3-C.sub.8 cycloalkyl, optionally substituted with one or
more, the same or different, R.sup.a groups; or (b) any two of
R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 together
with the carbons to which they are attached form a C.sub.4-C.sub.8
cycloalkyl group, optionally substituted with one or more, the same
or different, R.sup.a groups, while the remaining R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are as defined in (a);
R.sup.a is hydrogen, deuterium, CD.sub.3, C.sub.1-C.sub.6 alkyl,
OH, halogen, CN, CF.sub.3, O--C.sub.1-C.sub.6 alkyl, O-aryl,
O-heteroaryl, O--C.sub.3-C.sub.8 cycloalkyl, O-heterocyclyl,
--NR.sup.bR.sup.b, --COOR.sup.b or --CONR.sup.bR.sup.b; R.sup.b is
independently at each occurrence, the same or different, hydrogen,
linear or branched C.sub.1-C.sub.12 alkyl, aryl, heteroaryl,
heterocyclyl or C.sub.3-C.sub.8 cycloalkyl; X.sup.1 and X.sup.2 are
independently O, S or NR.sup.c; Y is O, S or NR.sup.c; R.sup.c is
hydrogen; m is 0, 1 or 2, and Nu is a nucleoside; provided that
either the carbon bearing the R.sup.1 and R.sup.2 groups, the
carbon bearing the R.sup.3 and R.sup.4 groups, or both, is chiral;
wherein the compound of formula (IV) is at least 90%
stereochemically pure, with the proviso that Compound IV does not
include the following compounds: ##STR00520##
15. (canceled)
16. The compound according to claim 14, having any one of formulae
(V)-(Vc): ##STR00521## wherein (a) R.sup.1, R.sup.3, R.sup.5 and
R.sup.6 are independently hydrogen, CD.sub.3, CF.sub.3, linear or
branched C.sub.1-C.sub.20 alkyl, optionally substituted with one or
more, the same or different, R.sup.a groups; linear or branched
C.sub.2-C.sub.12 alkenyl; optionally substituted with one or more,
the same or different, R.sup.a groups; linear or branched
C.sub.2-C.sub.12 alkynyl, optionally substituted with one or more,
the same or different, R.sup.a groups; aryl, optionally substituted
with one or more, the same or different, R.sup.a groups;
heteroaryl, optionally substituted with one or more, the same or
different, R.sup.a groups; heterocyclyl, optionally substituted
with one or more, the same or different, R.sup.a groups; or
C.sub.3-C.sub.8 cycloalkyl, optionally substituted with one or
more, the same or different, R.sup.a groups; or (b) any two of
R.sup.1, R.sup.3, R.sup.5, and R.sup.6 together with the carbons to
which they are attached form a C.sub.4-C.sub.8 cycloalkyl group,
optionally substituted with one or more, the same or different,
R.sup.a groups, while the R.sup.1, R.sup.3, R.sup.5 and R.sup.6
remaining are as defined above in (a); R.sup.a is hydrogen,
deuterium, CD.sub.3, C.sub.1-C.sub.6 alkyl, OH, halogen, CN,
CF.sub.3, O--C.sub.1-C.sub.6 alkyl, O-aryl, O-heteroaryl,
O--C.sub.3-C.sub.8 cycloalkyl, O-heterocyclyl, --NR.sup.bR.sup.b,
--COOR.sup.b or --CONR.sup.bR.sup.b; R.sup.b is independently at
each occurrence, the same or different, hydrogen, linear or
branched C.sub.1-C.sub.12 alkyl, aryl, heteroaryl, heterocyclyl or
C.sub.3-C.sub.8 cycloalkyl; Y is O, S or NR.sup.c, wherein R.sup.c
is hydrogen or C.sub.1-C.sub.4 alkyl; m is 0, 1, or 2, and Nu is a
nucleoside.
17. (canceled)
18. The compound of claim 14 of the formula ##STR00522## wherein
R.sup.2 and R.sup.4 are independently hydrogen, CD.sub.3 or
CF.sub.3, linear or branched C.sub.1-C.sub.20 alkyl, optionally
substituted with one or more, the same or different, R.sup.a
groups; linear or branched C.sub.2-C.sub.12 alkenyl; optionally
substituted with one or more, the same or different, R.sup.a
groups; linear or branched C.sub.2-C.sub.12 alkynyl, optionally
substituted with one or more, the same or different, R.sup.a
groups; aryl, optionally substituted with one or more, the same or
different, R.sup.a groups; heteroaryl, optionally substituted with
one or more, the same or different, R.sup.a groups; heterocyclyl,
optionally substituted with one or more, the same or different,
R.sup.a groups; or C.sub.3-C.sub.8 cycloalkyl, optionally
substituted with one or more, the same or different, R.sup.a
groups; R.sup.a is hydrogen, deuterium, CD.sub.3, C.sub.1-C.sub.6
alkyl, OH, halogen, CN, CF.sub.3, O--C.sub.1-C.sub.6 alkyl, O-aryl,
O-heteroaryl, O--C.sub.3-C.sub.8 cycloalkyl, O-heterocyclyl,
--NR.sup.bR.sup.b, --COOR.sup.b or --CONR.sup.bR.sup.b; R.sup.b is
independently at each occurrence, the same or different, hydrogen,
linear or branched C.sub.1-C.sub.12 alkyl, aryl, heteroaryl,
heterocyclyl or C.sub.3-C.sub.8 cycloalkyl; R.sup.7 is hydrogen;
CD.sub.3, OH, halogen, CN, CF.sub.3, linear or branched
C.sub.1-C.sub.6 alkyl; linear or branched C.sub.2-C.sub.6 alkenyl,
or linear or branched C.sub.2-C.sub.6 alkynyl; X.sup.1 and X.sup.2
are independently O, S or NR.sup.c; Y is O, S or NR.sup.c; R.sup.c
is hydrogen or C.sub.1-C.sub.4 alkyl; n is 0, 1, 2, 3 or 4; and Nu
is a nucleoside.
19. (canceled)
20. The compound according to claim 18 having the formula:
##STR00523## wherein R.sup.2 and R.sup.4 are independently
hydrogen, CD.sub.3, CF.sub.3, linear or branched C.sub.1-C.sub.20
alkyl, optionally substituted with one or more, the same or
different, R.sup.a groups; linear or branched C.sub.2-C.sub.12
alkenyl; optionally substituted with one or more, the same or
different, R.sup.a groups; linear or branched C.sub.2-C.sub.12
alkynyl, optionally substituted with one or more, the same or
different, R.sup.a groups; aryl, optionally substituted with one or
more, the same or different, R.sup.a groups; heteroaryl, optionally
substituted with one or more, the same or different, R.sup.a
groups; heterocyclyl, optionally substituted with one or more, the
same or different, R.sup.a groups; or C.sub.3-C.sub.8 cycloalkyl,
optionally substituted with one or more, the same or different,
R.sup.a groups; R.sup.a is hydrogen, deuterium, CD.sub.3,
C.sub.1-C.sub.6 alkyl, OH, halogen, CN, CF.sub.3,
O--C.sub.1-C.sub.6 alkyl, O-aryl, O-heteroaryl, O--C.sub.3-C.sub.8
cycloalkyl, O-heterocyclyl, --NR.sup.bR.sup.b, --COOR.sup.b or
--CONR.sup.bR.sup.b; R.sup.b is independently at each occurrence,
the same or different, hydrogen, linear or branched
C.sub.1-C.sub.12 alkyl, aryl, heteroaryl, heterocyclyl or
C.sub.3-C.sub.8 cycloalkyl; provided that either the carbon bearing
the R.sup.1 or R.sup.2 groups, the carbon bearing the R.sup.3 or
R.sup.4 groups, or both, is chiral; X.sup.1 and X.sup.2 are
independently O, S or NR.sup.c; Y is O, S or NR.sup.c; R.sup.c is
hydrogen or C.sub.1-C.sub.4 alkyl; and Nu is a nucleoside.
21. A method of making the compound of claim 1, comprising reacting
a compound of formula (VII): ##STR00524## wherein X and Y are
independently O or S, and Ph is phenyl, optionally substituted with
on one or more groups chosen from linear or branched
C.sub.1-C.sub.6 alkyl, aryl, heteroaryl, halogen, CN, or NO.sub.2,
with an epoxide or an episulfide to form the compound of claim
1.
22. The method of claim 21, wherein the epoxide has the following
formula (VIII): ##STR00525## wherein (a) R.sup.1, R.sup.2, R.sup.3
and R.sup.4 are independently hydrogen, CD.sub.3, CF.sub.3, linear
or branched C.sub.1-C.sub.20 alkyl, optionally substituted with one
or more, the same or different, R.sup.a groups; linear or branched
C.sub.2-C.sub.12 alkenyl; optionally substituted with one or more,
the same or different, R.sup.a groups; linear or branched
C.sub.2-C.sub.12 alkynyl, optionally substituted with one or more,
the same or different, R.sup.a groups; aryl, optionally substituted
with one or more, the same or different, R.sup.a groups;
heteroaryl, optionally substituted with one or more, the same or
different, R.sup.a groups; heterocyclyl, optionally substituted
with one or more, the same or different, R.sup.a groups; or
C.sub.3-C.sub.8 cycloalkyl, optionally substituted with one or
more, the same or different, R.sup.a groups; or (b) any two of
R.sup.1, R.sup.2, R.sup.3, and R.sup.4 together with the carbons to
which they are attached form C.sub.4-C.sub.8 cycloalkyl, optionally
substituted with one or more, the same or different, R.sup.a
groups, while the remaining R.sup.1, R.sup.2, R.sup.3, and R.sup.4
are as defined in (a); R.sup.a is hydrogen, deuterium, tritium;
halogen; linear or branched C.sub.1-C.sub.6 alkyl; OH, CN,
CF.sub.3, O--C.sub.1-C.sub.6 alkyl, O-aryl, O-heteroaryl,
O--C.sub.3-C.sub.8 cycloalkyl, O-heterocyclyl, --NR.sup.bR.sup.b,
--COOR.sup.b or --CONR.sup.bR.sup.b linear or branched
C.sub.2-C.sub.6 alkenyl, optionally substituted with linear or
branched C.sub.1-C.sub.6 alkyl; or linear or branched
C.sub.2-C.sub.6 alkynyl, optionally substituted with a linear or
branched C.sub.1-C.sub.6 alkyl; and R.sup.b is independently at
each occurrence, the same or different, hydrogen, linear or
branched C.sub.1-C.sub.12 alkyl, aryl, heteroaryl, heterocyclyl or
C.sub.3-C.sub.8 cycloalkyl; provided that either the carbon bearing
the R.sup.1 or R.sup.2 groups, the carbon bearing the R.sup.3 or
R.sup.4 groups, or both, is chiral.
23. (canceled)
24. (canceled)
25. (canceled)
26. A method of making the compound of claim 1, comprising reacting
PCl.sub.3 with an optionally substituted mercaptoethanol or
mercaptopropanol, wherein the mercaptoethanol or the
mercaptopropanol is chosen from the following formulae:
##STR00526## wherein (a) R.sup.1, R.sup.2, R.sup.3, R.sup.4,
R.sup.5 and R.sup.6 are independently hydrogen, CD.sub.3, CF.sub.3,
linear or branched C.sub.1-C.sub.20 alkyl, optionally substituted
with one or more, the same or different, R.sup.a groups; linear or
branched C.sub.2-C.sub.12 alkenyl; optionally substituted with one
or more, the same or different, R.sup.a groups; linear or branched
C.sub.2-C.sub.12 alkynyl, optionally substituted with one or more,
the same or different, R.sup.a groups; aryl, optionally substituted
with one or more, the same or different, R.sup.a groups;
heteroaryl, optionally substituted with one or more, the same or
different, R.sup.a groups; heterocyclyl, optionally substituted
with one or more, the same or different, R.sup.a groups; or
C.sub.3-C.sub.8 cycloalkyl, optionally substituted with one or
more, the same or different, R.sup.a groups; or (b) any two of
R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 together
with the carbons to which they are attached form a C.sub.4-C.sub.8
cycloalkyl group, optionally substituted with one or more, the same
or different, R.sup.a groups, while the remaining R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are as defined in (a);
R.sup.a is hydrogen, deuterium, tritium; halogen; linear or
branched C.sub.1-C.sub.6 alkyl; OH, CN, CF.sub.3,
O--C.sub.1-C.sub.6 alkyl, O-aryl, O-heteroaryl, O--C.sub.3-C.sub.8
cycloalkyl, O-heterocyclyl, --NR.sup.bR.sup.b, --COOR.sup.b or
--CONR.sup.bR.sup.b linear or branched C.sub.2-C.sub.6 alkenyl,
optionally substituted with linear or branched C.sub.1-C.sub.6
alkyl; or linear or branched C.sub.2-C.sub.6 alkynyl, optionally
substituted with a linear or branched C.sub.1-C.sub.6 alkyl; and
R.sup.b is independently at each occurrence, the same or different,
hydrogen, linear or branched C.sub.1-C.sub.12 alkyl, aryl,
heteroaryl, heterocyclyl or C.sub.3-C.sub.8 cycloalkyl; provided
that either the carbon bearing the R.sup.1 or R.sup.2 groups, the
carbon bearing the R.sup.3 or R.sup.4 groups, the R.sup.4 or
R.sup.5 groups or all of the above, is chiral.
27. A method for preparing the compound of claim 14, comprising
reacting the compound of claim 1 with a nucleoside in the presence
of a base.
28. (canceled)
29. (canceled)
30. (canceled)
31. A method of forming a phosphorothioate internucleoside linkage,
comprising reacting the compound of claim 14 with a nucleoside.
32. (canceled)
33. (canceled)
34. A method of making a cyclic dinucleotide, comprising: a)
reacting a compound of claim 1 with a nucleoside in the presence of
a base to form a loaded nucleoside; b) reacting the loaded
nucleoside with another nucleoside, thereby coupling the two
nucleosides; c) adding a compound of claim 1 to the coupled
nucleosides and reacting to form a cyclic dinucleotide.
35. A method of making an oligonucleotide, comprising: a) reacting
the compound of claim 1 with a first nucleoside in the presence of
a base to form a loaded nucleoside; b) reacting the loaded
nucleoside formed in step (a) with a second nucleoside, thereby
coupling the two nucleosides to form a dinucleotide; c) adding the
compound of claim 1 to form a loaded dinucleotide; d) adding
another nucleoside, thereby adding an additional nucleoside to said
dinucleotide; and e) repeating steps (c) and (d) one or more times
to form an oligonucleotide having a desired number of
nucleotides.
36. (canceled)
37. (canceled)
38. (canceled)
39. (canceled)
40. (canceled)
41. The compound according to claim 1 having the formula:
##STR00527##
42. (canceled)
43. The compound according to claim 1 of the formula:
##STR00528##
44. The compound according to claim 1 of the formula: ##STR00529##
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of U.S.
Provisional Application No. 62/657,551, filed on Apr. 13, 2018;
U.S. Provisional Application No. 62/668,098, filed on May 7, 2018;
U.S. Provisional Application No. 62/697,896, filed on Jul. 13,
2018; and U.S. Provisional Application No. 62/729,314, filed on
Sep. 10, 2018.
BACKGROUND OF THE INVENTION
Field of the Invention
[0002] The present invention relates to novel phosphorous (V)
(P(V)) reagents and methods for preparing enantiomerically enriched
(e.g., homochiral, optically pure, or single-isomer) p-chiral
organophosphorous compounds by using the novel (P(V)) reagents.
Background Art
[0003] Organophosphorous compounds have wide-ranging applications
as therapeutic and diagnostic agents, pest and insects control
agents, along with many other applications. Organophosphorous
compounds are generally classified based on the oxidation state of
the phosphorous atom: +5 (phosphorous (V)) or +3 (phosphorous
(III)). Organothiophosphates (phosphorous (V) compounds containing
sulfur attached to phosphorous) are a subclass of organophosphate
compounds where at least one of the oxygen atoms in the phosphate
is replaced by sulfur. In some situations, an asymmetry induced at
phosphorous results in chiral organothiophosphate compounds, which
makes this class of compounds particularly suitable in therapeutic,
diagnostic, research, and other applications.
[0004] A well-known and well-utilized example of
organothiophosphates are nucleic acids containing a thiophosphate,
e.g., phosphorothioate, backbone. The use of poly(nucleic acids),
for example oligonucleotides, containing a natural phosphodiester
backbone of DNA or RNA is limited by their instability to
nucleases. Phosphorothioate oligonucleotides are oligonucleotides
in which one of the non-crosslinked oxygen atoms in the
phosphodiester bond is replaced with a sulfur atom.
Oligonucleotides containing a phosphorothioate backbone have higher
nuclease resistance and cell membrane permeability compared with
oligonucleotides having a phosphodiester backbone.
[0005] Because of the chiral nature of a phosphorus atom in some
organothiophosphates, two kinds of stereoisomers (Rp- and
Sp-isomers) can exist. Therefore, in a P-chiral thiophosphate
derived oligonucleotide, thousands of diastereoisomers of compound
are possible, resulting in significant issues for development of
such agents. It is known that properties of oligonucleotides,
including binding affinity, sequence specific binding to
complementary RNA, and stability to nucleases, are affected by the
configurations at the phosphorous atoms. Further, it has been
suggested that homochiral isomers may have differential properties
(solubility, stability, activity, pharmacokinetics, etc.).
Therefore, it is highly desirable to prepare phosphorothioate
oligonucleotides with specific stereochemical configurations.
[0006] P-chiral organothiophosphates find another utility in making
bioconjugates, including protein- and peptide-nucleic acid
conjugates. Bioconjugates are compounds prepared through the
attachment of a therapeutic molecule, such as a small molecular
drug or a macromolecular drug to a functional molecule, such as a
lipid or a polymeric carrier, via a direct covalent bond or
covalently using a chemical linker. A typical bioconjugate
typically includes three basic building blocks: (1) a carrier
molecule, such as a polymer, a lipid, a peptide, or a protein; (2)
a therapeutic agent, including both small molecule chemicals and
macromolecule drugs; and (3) a chemical linker. Bioconjugates are
often used as a drug delivery strategy. Modifying therapeutic
agents with carrier molecules provides several advantages,
including, but not limited to, (1) optimization of physico-chemical
properties, (2) enhancing disease-specific targeting, (3) reducing
toxicity, and (4) controlling drug release profile. See Li, F. et
al., "Bioconjugate Therapeutics: Current Progress and Future
Perspective," Mol Pharm. 2017 May 1; 14(5): 1321-1324. For example,
properties of oligonucleotides and therapeutic agent, including
cell-specific delivery, target specificity, and cellular uptake
efficacy, can be manipulated by conjugating the agents to be
delivered to a peptide. Often times, it is desirable to use
thiophosphate-based linkages. Similarly, antibody-drug conjugates
(ADCs) often benefit from the presence of a single isomer
thiophosphate linkage. Moreover, organothiophosphates can be used
to make prodrugs, as well as novel linkers for induced protein
degradation.
[0007] Yet another use of organothiophosphate-based linkages is
incorporation of such linkages into cyclic dinucleotides (CDNs).
CDNs are important secondary signaling molecules in bacteria and in
mammalian cells. In the former case, they represent secondary
messengers affecting numerous responses of the prokaryotic cell,
whereas in the latter, they act as agonists of the innate immune
response. New discoveries have linked these two patterns of CDN
utilization as secondary messengers and have revealed unexpected
influences they have on shaping human genetic variation. Recently,
the use of CDNs as immune stimulating agents for the treatment of
cancer has received significant attention. Incorporation of
thiophosphate internucleoside linkages into a CDN confers
beneficial properties to the CDN.
[0008] However, there are difficulties associated with preparing
P(V) organothiophosphates and phosphodiester compounds. First, it
is difficult to prepare organophosphorous compounds using
phosphorous (111) chemistry. Thus, there is a need for a simple and
convenient method for linking molecules together through a
phosphorous (V) linkage.
[0009] Second, it is difficult to prepare organothiophosphates
where the stereochemical configuration at the phosphorus atom is
controlled. For example, the current approach to preparing single
isomer oligonucleotides, developed by Wada (FIG. 1), uses monomeric
units containing a 5'-protected nucleoside incorporated into a
chiral phosphoramidite. The Wada method usually produces modest
yields (41-75%) and low rates of reaction, driving the need for the
use of a significant excess of the phosphoramidite. In the first
step of the elongation cycle (1), the nitrogen is activated for
displacement through the use of an activator,
N-(cyanomethyl)-pyrrolidinium triflate, (a hygrosopic solid) and
then the solid supported 5'-hydroxyl can displace. The identity of
the activator is critical. The activator was designed to be acidic
enough to activate the nitrogen, but non-nucleophilic enough to
attack the resultant protonated phosphoramidite and scramble the
stereochemistry at phosphorous. In Wada's cases, the yields
(96-97%) and stereoselectivities (>99:1) of these couplings are
high. In the second step (2), the P(III) compound is oxidized with
(Me.sub.2NC(S)--S).sub.2 (DTD), then the pyrrolidine moiety of the
auxiliary is capped with trifluoroacylimidazole. The auxiliary and
DMTr group are finally cleaved with dichloroacetic acid (3). While
this works well on smaller fragments, when fragments get long (at
least 4-10mers), the generality of the activator disappears, and a
new activator must be identified through experimentation.
Additionally, the complexity of the process becomes a challenge for
making larger oligonucleosides.
[0010] Therefore, there remains a need for simple and selective
including regio- or stereo-selective synthetic methods of making
organothiophosphate or organophosphate-based molecules, including
oligonucleotides, peptide-oligonucleotide conjugates, ADCs,
prodrugs and CDNs.
BRIEF SUMMARY OF THE INVENTION
[0011] One aspect of the present disclosure is directed to methods
of making stereo-defined organosphosphorous (V) compounds using
novel chiral oxathiaphospholane sulfides, referred to herein as
"Compounds of the Disclosure," featuring a tetrahedral
phosphorous(V) (i.e., P(V)) center. More particularly, the methods
comprise coupling Compounds of the Disclosure represented by any
one of Formulae (I)-(IIIe), below, to a nucleophile, followed by a
reaction with a second nucleophile, thereby making a stereo-defined
organophoshorous (V) linkage between the two nucleophiles. Examples
of suitable nucleophiles include, but are not limited to, water,
hydroxide anions, alcohols, alkoxide anions, carboxylate anions,
thiols, thiolate anions, anions of a thiocarboxylic acids, amines,
and amides. To be suitable for the process of the present
disclosure, Compounds of the Disclosure are oxathiaphospholane
sulfide compounds that are designed to undergo a cleavage
(immolation) event upon (or after) addition of a second
nucleophile. Suitable steps for cleavage include, but are not
limited to, a ring strain/pinacol-induced cleavage, displacement of
a ring-atom followed by internal displacement (immolation) or a
strategic cyclic heteroatom in situ cleavage.
[0012] Compounds of the Disclosure are represented by any one of
Formulae (I)-(IIIe), wherein R.sup.1, R.sup.2, R.sup.1, R.sup.4,
R.sup.5, R.sup.6, X.sup.1, X.sup.2, Y, LG, n, and m are as defined
below:
##STR00001##
[0013] Compounds of the Disclosure are useful in preparing
organophosphorous (V) compounds, such as phosphate esters (e.g.,
phosphorothioates, phosphorodithioates, phosphodiesters) and
phosphate amides. Examples of organophosphorous (V) compounds that
can be prepared using Compounds of the Disclosure include, but are
not limited to, achiral and chiral thiophosphates relevant to CDNs
and antisense oligonucleotides, including CDNs and oligonucleotides
containing phosphorothiolate internucleoside linkages,
peptide-oligonucleotide conjugates, ADCs, phosphorylated natural
products, phosphorylated peptides, and organic molecules containing
a phosphorylation.
[0014] In another aspect, the present disclosure provides methods
of making Compounds of the Disclosure, comprising reacting a
dithioate salt with an epoxide or an episulfide disclosed herein.
In one embodiment, an epoxide is used. A suitable epoxide is
limonene oxide. In another embodiment, an episulfide is used. A
suitable episulfide is thiirane.
[0015] In another aspect, the present disclosure provides methods
of making Compounds of the Disclosure, comprising reacting
PCl.sub.3 with a mercaptoethanol or a mercaptopropanol, wherein the
mercaptoethanol or mercaptopropanol is chosen from the following
formulae:
##STR00002##
[0016] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 and
R.sup.6 are defined below.
[0017] In another embodiment, Compounds of the Disclosure of
Formulae (I)-(IIIe) can be made according to the following
exemplary reaction scheme:
##STR00003##
[0018] wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 and LG are as
defined below.
[0019] In another aspect, the present disclosure provides methods
of making Compounds of the Disclosure, comprising reacting a
P(O)Cl.sub.3 with a mercaptopropanol, wherein the mercaptopropanol
is represented by
##STR00004##
[0020] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 and
R.sup.6 are defined below.
[0021] Also provided are compounds represented by any one of
Formulae (IV)-(VIe), wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4,
R.sup.5, R.sup.6, X.sup.1, X.sup.2, Y, and Nu are as defined below,
also collectively referred to herein as "Compounds of the
Disclosure:"
##STR00005##
[0022] Compounds represented by any one of Formulae (IV)-(VIe) are
products of a reaction between compounds represented by any one of
Formulae (I)-(IIIe) and a nucleophile, which can be a nucleoside.
In this aspect, the 2'-hydroxy, 3'-hydroxy, or 5'-hydroxy of a
nucleoside acts as the nucleophile that reacts with the compounds
represented by any one of Formulae (I)-(IIIe). In some embodiments,
the nucleophile is an organic molecule containing a nucleophilic
atom. In another embodiment the nucleophilie can be a nucleoside.
In another embodiment, the nucleoside is a ribonucleoside. In
another embodiment, the nucleoside is a deoxyribonucleoside. In
some embodiments, the nucleoside comprises a naturally-occurring
nucleobase and/or sugar. In other embodiments, the nucleoside
comprises a modified nucleobase and/or sugar. In some embodiments,
the reaction is conducted in the presence of a base. Suitable bases
are 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),
2-tert-butyl-1,1,3,3-tetramethylguanidine (BTMG),
1,1,3,3-tetramethylguanidine (TMG), lithium
bis(trimethylsilyl)amide (LiHMDS), lithium tert-butoxide (LiOtBu),
potassium bis(trimethylsilyl)amide (KHMDS), potassium tert-butoxide
(KOtBu), sodium bis(trimethylsilyl)amide (NaHMDS), sodium
tert-butoxide (NaOtBu), 1,4-diazabicyclo[2.2.2]octane (DABCO),
1-methylimidazole (NMI), diisopropylethylamine (DIPEA), pyridine
(Pyr), 2,6-lutidine (2,6-Lut), and imidazole.
[0023] In another aspect, the present disclosure provides methods
of forming a thiophosphate internucleoside linkage, comprising
reacting the compound represented by any one of Formulae (IV)-(VIe)
with a nucleophile. Examples of nucleophile include, but are not
limited to, nucleophilic residues of nucleosides, peptides, drugs,
etc. In one embodiment, the nucleophile is a nucleoside. In one
embodiment, the linkage is a phosphorothioate internucleoside
linkage. In some embodiments, the nucleoside is a ribonucleoside.
In other embodiments, the nucleoside is a deoxyribonucleoside. In
some embodiments, the nucleoside comprises a naturally-occurring
nucleobase and/or sugar. In other embodiments, the nucleoside
comprises a modified nucleobase and/or sugar.
[0024] Also provided are methods of making a cyclic dinucleotide,
comprising:
[0025] a) reacting any one of Compounds of the Disclosure of
Formulae (I)-(IIIe) with a nucleoside in the presence of a
base;
[0026] b) reacting the compound formed in step (a) with another
nucleoside, thereby coupling the two nucleosides; and
[0027] c) adding any one of Compounds of the Disclosure of Formulae
(I)-(IIIe). In some embodiments, the method further comprises
deprotection of the nucleosides after step (b). In other
embodiments, the method further comprises another deprotection step
after step (c) to cyclize a precursor to form a CDN.
[0028] In some embodiments, the nucleoside is a ribonucleoside. In
other embodiments, the nucleoside is a deoxyribonucleoside. In some
embodiments, the nucleoside comprises a naturally-occurring
nucleobase and/or sugar. In other embodiments, the nucleoside
comprises a modified nucleobase and/or sugar. Suitable bases for
step (a) are as described above.
[0029] In one embodiment, one or both nucleosides comprise a
protecting group at the 5'-hydroxy. Useful protecting groups are
disclosed below. In one embodiment, the protecting group is
dimethoxytrityl (DMTr). In one embodiment, one or both nucleosides
comprise a protecting group at the 3'-hydroxy. In one embodiment,
the protecting group is TBS. In another embodiment, when the
nucleoside is a deoxyribonucleoside, it can comprise a protecting
group at the 2'-hydroxy. In one embodiment, the protecting group is
TOM. In another embodiment, the protecting group is TBDPS. Yet in
other embodiments, one or both nucleosides comprise a protecting
group on a base. In one embodiment, the protecting group is a
nucleophile protecting group. In one embodiment, the protecting
group is acetyl (Ac), isobutyl (iBu), dimethylformamidyl (DMF),
benzoyl (Bz), or benzyl (Bn). In another embodiment, neither
nucleoside comprises a protecting group on a base.
[0030] In another aspect, the present disclosure provides methods
of making an oligonucleotide, comprising:
[0031] a) reacting any one of Compounds of the Disclosure of
Formulae (I)-(IIIe) with a nucleoside in the presence of a
base;
[0032] b) reacting the compound formed in step (a) with another
nucleoside, thereby coupling the two nucleosides;
[0033] c) adding any one of Compounds of the Disclosure of Formulae
(I)-(IIIe);
[0034] d) adding another nucleoside, thereby coupling said
nucleoside to the growing oligonucleotide; and
[0035] e) repeating steps (c) and (d) until the oligonucleotide
comprises a desired number of nucleotides.
[0036] In some embodiments, the nucleoside is a ribonucleoside. In
other embodiments, the nucleoside is a deoxyribonucleoside. In some
embodiments, the nucleoside comprises a naturally-occurring
nucleobase and/or sugar. In other embodiments, the nucleoside
comprises a modified nucleobase and/or sugar.
[0037] In one embodiment, one, some or all nucleosides comprise a
protecting group at the 5'-hydroxy. In one embodiment, the
protecting group is DMTr. In one embodiment, one, some or all
comprises a protecting group at the 3'-hydroxy. In one embodiment,
the protecting group is tert-butyldimethylsilyl (TBS). In another
embodiment, when the nucleoside is a deoxyribonucleoside, it can
comprise a protecting group at the 2'-hydroxy. In one embodiment,
the protecting group is TOM. In another embodiment, the protecting
group is TBDPS. Yet in other embodiments, one, some or all
nucleosides comprise a protecting group on a base. In one
embodiment, the protecting group is Ac, iBu, DMF, Bn, or Bz. In
another embodiment, none of the nucleosides comprise a protecting
group on a base.
[0038] In some embodiments, the protecting group is removed after
each coupling step. In one embodiment, the nucleoside in step (a)
is attached to a resin at the 3'-end. In some embodiments, the
method comprising cleaving the oligonucleotide from the resin.
[0039] In some embodiments, achiral phosphate or dithiophosphate,
or chiral thiophosphate linkages between two nucleophilic
molecules, added sequentially, can be formed. This may be used to
link two molecules of interest, or in other applications, create
pro-drugs, or use in pharmaceutical applications.
[0040] Additional embodiments and advantages of the disclosure will
be set forth, in part, in the description that follows, and will
flow from the description, or can be learned by practice of the
disclosure. The embodiments and advantages of the disclosure will
be realized and attained by means of the elements and combinations
particularly pointed out in the appended claims.
[0041] It is to be understood that both the foregoing summary and
the following detailed description are exemplary and explanatory
only, and are not restrictive of the invention as claimed.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0042] FIG. 1 is a schematic of the Wada synthesis of
oligonucleotides with stereo-defined phosphorothioate linkages.
[0043] FIG. 2 is an example of a solid-phase synthesis of an
oligonucleotide in accordance with a method of the present
disclosure.
DETAILED DESCRIPTION OF THE INVENTION
Compounds of the Disclosure
[0044] Compounds of the Disclosure are phosphorous-containing
heterocycles containing phosphorous in (V) oxidation state (i.e.,
P(V)).
a. Phosphorous (V) Reagents
[0045] In one embodiment, Compounds of the Disclosure are compounds
represented by Formula (I):
##STR00006##
[0046] wherein
[0047] (a) R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6
are independently hydrogen, CD.sub.3 or CF.sub.3, linear or
branched C.sub.1-C.sub.20 alkyl, optionally substituted with one or
more, the same or different, R.sup.a groups; linear or branched
C.sub.2-C.sub.12 alkenyl; optionally substituted with one or more,
the same or different, R.sup.a groups; linear or branched
C.sub.2-C.sub.12 alkynyl, optionally substituted with one or more,
the same or different, R.sup.a groups; aryl, optionally substituted
with one or more, the same or different, R.sup.a groups;
heteroaryl, optionally substituted with one or more, the same or
different, R.sup.a groups; heterocyclyl, optionally substituted
with one or more, the same or different, R.sup.a groups; or
C.sub.3-C.sub.8 cycloalkyl, optionally substituted with one or
more, the same or different, R.sup.a groups;
[0048] or
[0049] (b) any two of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
and R.sup.6 together with the carbons to which they are attached
form a C.sub.4-C.sub.8 cycloalkyl group, optionally substituted
with one or more, the same or different, R.sup.a groups, while the
remaining R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6
are as defined above;
[0050] wherein
[0051] R.sup.a is hydrogen, deuterium, CD.sub.3, C.sub.1-C.sub.6
alkyl, OH, halogen, CN, CF.sub.3, O--C.sub.1-C.sub.6 alkyl, O-aryl,
O-heteroaryl, O--C.sub.3-C.sub.8 cycloalkyl, O-heterocyclyl,
--NR.sup.bR.sup.b, --COR.sup.b or --CONR.sup.bR.sup.b;
[0052] R.sup.b is independently at each occurrence, the same or
different, hydrogen, linear or branched C.sub.1-C.sub.12 alkyl,
aryl, heteroaryl, heterocyclyl or C.sub.3-C.sub.8 cycloalkyl;
[0053] X.sup.1 and X.sup.2 are independently O, S or NR.sup.c;
wherein R.sup.c is hydrogen or C.sub.1-C.sub.4 alkyl,
[0054] Y is O, S or NR.sup.c;
[0055] m is 0, 1 or 2, and
[0056] LG is a leaving group.
[0057] In one embodiment, the carbon bearing the R.sup.1 and
R.sup.2 groups, the carbon bearing the R.sup.3 and R.sup.4 groups,
or both, in the compound of formula (I) is chiral, and the compound
of formula (I) is at least 90% stereochemically pure.
[0058] In another embodiment, the compounds of the present
disclosure are represented by Formula (I), wherein
[0059] (a) R.sup.1, R.sup.2, R.sup.3, R.sup.4, R, and R.sup.6 are
independently hydrogen; linear or branched C.sub.1-C.sub.3 alkyl;
aryl; heteroaryl; or C.sub.3-C.sub.6 cycloalkyl;
[0060] or
[0061] (b)) any two of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
and R.sup.6 together with the carbons to which they are attached
form C.sub.4-C.sub.6 cycloalkyl, while the remaining R.sup.1,
R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are independently
hydrogen; linear or branched C.sub.1-C.sub.3 alkyl; aryl;
heteroaryl; or C.sub.3-C.sub.6 cycloalkyl;
[0062] provided that either the carbon bearing the R.sup.1 or
R.sup.2 groups, the carbon bearing the R.sup.3 or R.sup.4 groups,
or both, is chiral;
[0063] X.sup.1 and X.sup.2 are independently O, S, or NR.sup.c,
wherein R.sup.c is hydrogen or C.sub.1-C.sub.4 alkyl;
[0064] Y is O, S or NR.sup.c;
[0065] m is 0, 1 or 2, and
[0066] LG is a leaving group,
[0067] and the compound of formula (I) is at least 90%
stereochemically pure.
[0068] In one embodiment, Compounds of the Disclosure are compounds
represented by Formula (Ia):
##STR00007##
[0069] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, X.sup.1,
X.sup.2, Y, and LG are as defined above.
[0070] In some embodiments, X.sup.1 is S. and X.sup.2 is O. In
other embodiments, X.sup.1 is O, and X.sup.2 is S. In other
embodiments, both X.sup.1 and X.sup.2 are S. In some embodiments, Y
is O or S. In one embodiment, Y is O. In another embodiment, Y is
S. In another embodiment, Y is O or S. In one embodiment, X.sup.1
is S, X.sup.2 is O, and Y is O. In another embodiment, X.sup.1 is
S, X.sup.2 is O, and Y is S. In one embodiment, X.sup.1 is O,
X.sup.2 is S, and Y is O. In another embodiment, X.sup.1 is O,
X.sup.2 is S, and Y is S. In one embodiment. X.sup.1 is S, X.sup.2
is S, and Y is S.
[0071] When both X.sup.1 and X.sup.2 are sulfur, Compounds of the
Disclosure do not have phosphorus stereochemistry.
[0072] In another embodiment, Compounds of the Disclosure are
compounds represented by Formulae (II-IIc):
##STR00008##
[0073] wherein
[0074] (a) R.sup.1, R.sup.3, R.sup.5 and R.sup.6 are independently
hydrogen, CD.sub.3 or CF.sub.3, linear or branched C.sub.1-C.sub.20
alkyl, optionally substituted with one or more, the same or
different, R.sup.a groups; linear or branched C.sub.2-C.sub.12
alkenyl; optionally substituted with one or more, the same or
different, R.sup.a groups; linear or branched C.sub.2-C.sub.12
alkynyl, optionally substituted with one or more, the same or
different, R.sup.a groups; aryl, optionally substituted with one or
more, the same or different, R.sup.a groups; heteroaryl, optionally
substituted with one or more, the same or different, R.sup.a
groups; heterocyclyl, optionally substituted with one or more, the
same or different. R.sup.a groups; or C.sub.3-C.sub.8 cycloalkyl,
optionally substituted with one or more, the same or different,
R.sup.a groups;
[0075] or
[0076] (b) any two of R.sup.1, R.sup.3, R.sup.5, and R.sup.6
together with the carbons to which they are attached form a
C.sub.4-C.sub.8 cycloalkyl group, optionally substituted with one
or more, the same or different, R.sup.a groups, while the R.sup.1,
R.sup.3, R.sup.5 and R.sup.6 are independently are as defined in
(a); and
[0077] R.sup.a is hydrogen; deuterium, CD.sub.3, C.sub.1-C.sub.6
alkyl, OH, halogen, CN, CF.sub.3, O--C.sub.1-C.sub.6 alkyl, O-aryl,
--O-heteroaryl, -heteroaryl, O--C.sub.3-C.sub.8 cycloalkyl,
O-heterocyclyl, --NR.sup.bR.sup.b, --COOR.sup.b or
--CONR.sup.bR.sup.b;
[0078] R.sup.b is independently, at each occurrence, the same or
different, hydrogen, linear or branched C.sub.1-C.sub.12 alkyl,
aryl, heteroaryl, heterocyclyl or C.sub.3-C.sub.8 cycloalkyl;
[0079] Y is O, S or NR.sup.c, where R.sup.c is hydrogen or
C.sub.1-C.sub.4 alkyl;
[0080] m is 0, 1 or 2, and
[0081] LG is a leaving group.
[0082] In one embodiment, the compounds of the present disclosure
are represented by Formulae (II-IIc), wherein
[0083] (a) R.sup.1, R.sup.3, R.sup.5 and R.sup.6 are independently
hydrogen; linear or branched C.sub.1-C.sub.3 alkyl; C.sub.1-C.sub.3
alkyl substituted with halogen; CN; aryl; heteroaryl; or
C.sub.3-C.sub.6 cycloalkyl;
[0084] or
[0085] (b) any two of R.sup.1, R.sup.3, R.sup.5, and R.sup.6
together with the carbons to which they are attached form
C.sub.4-C.sub.6 cycloalkyl, while the R.sup.1, R.sup.3, R.sup.5 and
R.sup.6 are independently are as defined in (a);
[0086] provided that either the carbon bearing the R.sup.1 and
R.sup.2 groups, the carbon bearing the R.sup.3 and R.sup.4 groups,
or both, is chiral;
[0087] Y is O, S or NR.sup.c, wherein R.sup.c is hydrogen or
C.sub.1-C.sub.4 alkyl;
[0088] m is 0, 1, or 2, and
[0089] LG is a leaving group.
[0090] In one embodiment, Compounds of the Disclosure are compounds
represented by Formulae (IId-IIg):
##STR00009##
[0091] wherein
[0092] (a) R.sup.1 and R.sup.3 are independently hydrogen, CD.sub.3
or CF.sub.3, linear or branched C.sub.1-C.sub.20 alkyl, optionally
substituted with one or more, the same or different, R.sup.a
groups; linear or branched C.sub.2-C.sub.12 alkenyl; optionally
substituted with one or more, the same or different, R.sup.a
groups; linear or branched C.sub.2-C.sub.12 alkynyl, optionally
substituted with one or more, the same or different, R.sup.a
groups; aryl, optionally substituted with one or more, the same or
different, R.sup.a groups; heteroaryl, optionally substituted with
one or more, the same or different, R.sup.a groups; heterocyclyl,
optionally substituted with one or more, the same or different.
R.sup.a groups; or C.sub.3-C.sub.8 cycloalkyl, optionally
substituted with one or more, the same or different, R.sup.a
groups;
[0093] or
[0094] (b) R.sup.1 and R.sup.3 together with the carbons to which
they are attached form a C.sub.4-C.sub.8 cycloalkyl group,
optionally substituted with one or more, the same or different,
R.sup.a groups;
[0095] R.sup.a is hydrogen; deuterium, CD.sub.3, C.sub.1-C.sub.6
alkyl, OH, halogen, CN, CF.sub.3, O--C.sub.1-C.sub.6 alkyl, O-aryl,
O-heteroaryl, O--C.sub.3-C.sub.8 cycloalkyl, O-heterocyclyl,
--NR.sup.bR.sup.b, --COOR.sup.b or --CONR.sup.bR.sup.b;
[0096] R.sup.b is independently at each occurrence, the same or
different, hydrogen, linear or branched C.sub.1-C.sub.12 alkyl,
aryl, heteroaryl, heterocyclyl or C.sub.3-C.sub.8 cycloalkyl;
[0097] provided that either the carbon bearing the R.sup.1 and
R.sup.2 groups, the carbon bearing the R.sup.3 and R.sup.4 groups,
or both, is chiral; and
[0098] LG is a leaving group.
[0099] In yet another embodiment, Compounds of the Disclosure are
compounds represented by Formulae (III-IIIc):
##STR00010##
[0100] wherein
[0101] R.sup.2 and R.sup.4 are independently hydrogen or linear or
branched C.sub.1-C.sub.6 alkyl, optionally substituted with one or
more, the same or different, R.sup.a groups; and
[0102] R.sup.a and LG are as defined above.
[0103] In one embodiment, Compounds of the Disclosure are compounds
represented by the following Formula (IIId):
##STR00011##
[0104] wherein
[0105] R.sup.2 and R.sup.4 are independently hydrogen, CD.sub.3 or
CF.sub.3, linear or branched C.sub.1-C.sub.20 alkyl, optionally
substituted with one or more, the same or different, R.sup.a
groups; linear or branched C.sub.2-C.sub.12 alkenyl; optionally
substituted with one or more, the same or different, R.sup.a
groups; linear or branched C.sub.2-C.sub.12 alkynyl, optionally
substituted with one or more, the same or different, R.sup.a
groups; aryl, optionally substituted with one or more, the same or
different, R.sup.a groups; heteroaryl, optionally substituted with
one or more, the same or different, R.sup.a groups; heterocyclyl,
optionally substituted with one or more, the same or different,
R.sup.a groups; or C.sub.3-C.sub.8 cycloalkyl, optionally
substituted with one or more, the same or different, R.sup.a
groups;
[0106] R.sup.a is hydrogen; deuterium, CD.sub.3, C.sub.1-C.sub.6
alkyl, OH, halogen, CN, CF.sub.3, O--C.sub.1-C.sub.6 alkyl, O-aryl,
O-heteroaryl, O--C.sub.3-C.sub.8 cycloalkyl, O-heterocyclyl,
--NR.sup.bR.sup.b, --COOR.sup.b or --CONR.sup.bR.sup.b;
[0107] R.sup.b is independently at each occurrence, the same or
different, hydrogen, linear or branched C.sub.1-C.sub.12 alkyl,
aryl, heteroaryl, heterocyclyl or C.sub.3-C.sub.8 cycloalkyl;
[0108] R.sup.7 is hydrogen; CD.sub.3, OH, halogen, CN, CF.sub.3,
linear or branched C.sub.1-C.sub.6 alkyl; linear or branched
C.sub.2-C.sub.6 alkenyl, or linear or branched C.sub.2-C.sub.6
alkynyl;
[0109] X.sup.1 and X.sup.2 are independently O, S or NR.sup.c,
wherein R.sup.c is hydrogen or C.sub.1-C.sub.4 alkyl;
[0110] Y is O, S or NR.sup.c;
[0111] n is 0, 1, 2, 3 or 4; and
[0112] LG is a leaving group.
[0113] In yet another embodiment, Compounds of the Disclosure are
compounds represented by Formula (IIIe):
##STR00012##
[0114] wherein
[0115] R.sup.2 and R.sup.4 are independently hydrogen, CD.sub.3 or
CF.sub.3, linear or branched C.sub.1-C.sub.20 alkyl, optionally
substituted with one or more, the same or different, R.sup.a
groups; linear or branched C.sub.2-C.sub.12 alkenyl; optionally
substituted with one or more, the same or different, R.sup.a
groups; linear or branched C.sub.2-C.sub.12 alkynyl, optionally
substituted with one or more, the same or different, R.sup.a
groups; aryl, optionally substituted with one or more, the same or
different, R.sup.a groups; heteroaryl, optionally substituted with
one or more, the same or different, R.sup.a groups; heterocyclyl,
optionally substituted with one or more, the same or different,
R.sup.a groups; or C.sub.3-C.sub.8 cycloalkyl, optionally
substituted with one or more, the same or different, R.sup.a
groups;
[0116] R.sup.a is hydrogen; deuterium, CD.sub.3, C.sub.1-C.sub.6
alkyl, OH, halogen, CN, CF.sub.3, O--C.sub.1-C.sub.6 alkyl, O-aryl,
O-heteroaryl, O--C.sub.3-C.sub.8 cycloalkyl, O-heterocyclyl,
--NR.sup.bR.sup.b, --COOR.sup.b or --CONR.sup.bR.sup.b;
[0117] R.sup.b is independently at each occurrence, the same or
different, hydrogen, linear or branched C.sub.1-C.sub.12 alkyl,
aryl, heteroaryl, heterocyclyl or C.sub.3-C.sub.8 cycloalkyl;
[0118] provided that either the carbon bearing the R.sup.1 and
R.sup.2 groups, the carbon bearing the R.sup.3 and R.sup.4 groups,
or both, is chiral;
[0119] X.sup.1 and X.sup.2 are independently O, S or NR.sup.c,
wherein R.sup.c is hydrogen or C.sub.1-C.sub.4 alkyl;
[0120] Y is O, S or NR.sup.c; and
[0121] LG is a leaving group.
[0122] In some embodiments, Compounds of the Disclosure can be
characterized by ring strain, as represented by the following
non-limiting examples:
##STR00013##
[0123] In additional embodiments, Compounds of the Disclosure
comprise a strategic heteroatom (which makes the compounds amenable
to in situ cleavage), as represented by the following non-limiting
examples:
##STR00014##
[0124] In another embodiment, Compounds of the Disclosure are any
one or more of the compounds of Table 1.
TABLE-US-00001 TABLE 1 Cpd. No. Structure Name 1-1 ##STR00015##
(4S,5R)-2-(4-nitrophenoxy)-4,5-diphenyl-1,3,2- oxathiaphospholane
2-sulfide 1-2 ##STR00016##
(4S,5S)-2-(4-nitrophenoxy)-4,5-diphenyl-1,3,2- oxathiaphospholane
2-sulfide 1-3 ##STR00017##
(3aR,6S,7aR)-2,3a-dimethyl-6-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2- sulfide 1-4
##STR00018## (3aR,6S,7aR)-2-(dodecylthio)-3a-methyl-6-
(prop-1-en-2- yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-
sulfide 1-5 ##STR00019##
N-((2S,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2-
yl)-2-sulfidohexahydro- benzo[d][1,3,2]oxathiaphosphol-
2-yl)nicotinamide 1-6 ##STR00020##
N-((3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2-yl)- 2-sulfidohexahydro-
benzo[d][1,3,2]oxathiaphosphol-
2-yl)-5H-dibenzo[b,f]azepine-5-carboxamide 1-7 ##STR00021##
1-((2R,4S,5S)-5-(((tert- butyldimethylsilyl)oxy)methyl)-4-
(((3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2-yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxa-
thiaphosphol-2-yl)amino)tetrahydrofuran-2-yl)-
5-methylpyrimidine-2,4(1H,3H)-dione 1-8 ##STR00022##
(4S,5R)-4,5-dimethyl-2-(4-nitrophenoxy)-1,3,2- oxathiaphospholane
2-sulfide 1-9 ##STR00023##
(4S,5S)-4,5-dimethyl-2-(4-nitrophenoxy)-1,3,2- oxathiaphospholane
2-sulfide 1-10 ##STR00024##
2-(4-nitrophenoxy)-1,3,2-oxathiaphosphinane 2-oxide 1-11
##STR00025## 2-((perfluorophenyl)thio)-1,3,2- oxathiaphosphinane
2-oxide 1-12 ##STR00026## (5R)-2-(4-nitrophenoxy)-5-phenyl-1,3,2-
oxathiaphospholane 2-sulfide 1-13 ##STR00027##
(2S,4S)-2-((perfluorophenyl)thio)-4-phenyl-
1,3,2-oxathiaphospholane 2-sulfide 1-14 ##STR00028##
(2R,4R)-2-((perfluorophenyl)thio)-4-phenyl-
1,3,2-oxathiaphospholane 2-sulfide 1-15 ##STR00029##
(5R)-5-methyl-2-(4-nitrophenoxy)-1,3,2- oxathiaphospholane
2-sulfide 1-16 ##STR00030##
(2R,3aS,5R,7aS)-7a-methyl-2-(4-nitrophenoxy)- 5-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2- sulfide 1-17
##STR00031## (2S,3aR,5R,7aR)-7a-methyl-2-(4-nitrophenoxy)-
5-(prop-1-en-2- yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-
sulfide 1-18 ##STR00032##
(2S,3aS,6R,7aR)-3a-methyl-2-(4-nitrophenoxy)- 6-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2- sulfide 1-19
##STR00033## (2R,3aR,6S,7aS)-3a-methyl-2-(4-nitrophenoxy)-
6-(prop-1-en-2- yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-
sulfide 1-20 ##STR00034## (3aS,7aS)-2-(4-
nitrophenoxy)hexahydrobenzo[d][1,3,2]oxa- thiaphosphole 2-sulfide
1-21 ##STR00035## (3aS,7aS)-2-(4-nitrophenoxy)hexahydro-
benzo[d][1,3,2]oxathiaphosphole 2-sulfide 1-22 ##STR00036##
(3aS,7aS)-7a-methyl-2-(4- nitrophenoxy)hexahydrobenzo[d][1,3,2]oxa-
thiaphosphole 2-sulfide 1-23 ##STR00037## (3aS,7aS)-3a-methyl-2-(4-
nitrophenoxy)hexahydrobenzo[d][1,3,2]oxa- thiaphosphole 2-sulfide
1-24 ##STR00038## (4S,5R)-5-methyl-2-(4-nitrophenoxy)-4-phenyl-
1,3,2-oxathiaphospholane 2-sulfide 1-25 ##STR00039##
(4R,5S)-4-methyl-2-(4-nitrophenoxy)-5-phenyl-
1,3,2-oxathiaphospholane 2-sulfide 1-26 ##STR00040##
(4S,5R)-2-(perfluorophenoxy)-4,5-diphenyl- 1,3,2-oxathiaphospholane
2-sulfide 1-27 ##STR00041##
(4S,5R)-2-(naphthalen-2-ylthio)-4,5-diphenyl-
1,3,2-oxathiaphospholane 2-sulfide 1-28 ##STR00042##
(4S,5R)-2-((4-nitrophenyl)thio)-4,5-diphenyl-
1,3,2-oxathiaphospholane 2-sulfide 1-29 ##STR00043##
(4S,5R)-2-((4-fluorophenyl)thio)-4,5-diphenyl-
1,3,2-oxathiaphospholane 2-sulfide 1-30 ##STR00044##
(4S,5R)-2-((4-fluorophenyl)thio)-4,5-diphenyl-
1,3,2-oxathiaphospholane 2-sulfide 1-31 ##STR00045##
(2S,3aS,5R,7aS)-7a-methyl-2-
((perfluorophenyl)thio)-5-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxa- thiaphosphole 2-sulfide 1-32
##STR00046## (2R,3aR,5S,7aR)-7a-methyl-2-
((perfluorophenyl)thio)-5-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 1-33
##STR00047## (2S,3aS,6R,7aR)-3a-methyl-2-
((perfluorophenyl)thio)-6-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 1-34
##STR00048## (2R,3aR,6S,7aS)-3a-methyl-2-
((perfluorophenyl)thio)-6-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 1-35
##STR00049## (4S,5R)-2-((perfluorophenyl)thio)-4,5-diphenyl-
1,3,2-oxathiaphospholane 2-sulfide 1-36 ##STR00050##
(4S,5R)-2-(4-nitrophenoxy)-4,5-diphenyl-1,3,2- oxathiaphospholane
2-oxide 1-37 ##STR00051##
(4S,5R)-4,5-dimethyl-2-(4-nitrophenoxy)-1,3,2- oxathiaphospholane
2-oxide 1-38 ##STR00052##
(3aS,5R,7aS)-7a-methyl-2-(4-nitrophenoxy)-5- (prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-oxide 1-39
##STR00053## (3aS,6R,7aR)-3a-methyl-2-(4-nitrophenoxy)-6-
(prop-1-en-2- yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-oxide
1-40 ##STR00054## (2R,3aS,5R,7aS)-7a-methyl-2-(4-nitrophenoxy)-
5-(prop-1-en-2- yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole
2-sulfide 1-41 ##STR00055## (2S,3aR,5R,7aR)-2-(4-bromophenoxy)-7a-
methyl-5-(prop-1-en-2- yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole
2-sulfide 1-42 ##STR00056## (2S,3aS,6R,7aR)-2-(4-bromophenoxy)-3a-
methyl-6-(prop-1-en-2- yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole
2-sulfide 1-43 ##STR00057## (2R,3aR,6S,7aS)-2-(4-bromophenoxy)-3a-
methyl-6-(prop-1-en-2- yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole
2-sulfide 1-44 ##STR00058## trimethylamine salt of
2-hydroxy-4,6-diphenyl- 1,3,2-oxathiaphosphinane 2-oxide 1-45
##STR00059## triethylamine salt of (4S,6R)-2-hydroxy-4,6-
diphenyl-1,3,2-oxathiaphosphinane 2-oxide 1-46 ##STR00060##
triethylamine salt of (4R,6R)-2-hydroxy-4,6-
diphenyl-1,3,2-oxathiaphosphinane 2-oxide 1-47 ##STR00061## sodium
(4S,6R)-4,6-diphenyl-1,3,2- oxathiaphosphinan-2-olate 2-oxide 1-48
##STR00062## sodium (4R,6R)-4,6-diphenyl-1,3,2-
oxathiaphosphinan-2-olate 2-oxide 1-49 ##STR00063##
2-chloro-4,6-diphenyl-1,3,2-oxathiaphosphinane 2-oxide 1-50
##STR00064## (4S,6R)-2-chloro-4,6-diphenyl-1,3,2-
oxathiaphosphinane 2-oxide 1-51 ##STR00065##
(2R,4R,6R)-2-chloro-4,6-diphenyl-1,3,2- oxathiaphosphinane 2-oxide
1-52 ##STR00066## (2R,4R,6S)-2-(4-nitrophenoxy)-4,6-diphenyl-
1,3,2-oxathiaphosphinane 2-oxide 1-53 ##STR00067##
(2R,4R,6R)-2-(4-nitrophenoxy)-4,6-diphenyl-
1,3,2-oxathiaphosphinane 2-oxide
[0125] In one embodiment, Compounds of the Disclosure are compounds
selected from the group consisting of compounds 1-1 to 1-9, 1-12,
and 1-15 to 1-43. In another embodiment, Compounds of the
Disclosure are compounds selected from the group consisting of
compounds 1-10, 1-11, 1-13, and 1-14. In another embodiment,
Compounds of the Disclosure are compounds selected from the group
consisting of compounds 1-44 to 1-53.
[0126] In yet another embodiment, Compounds of the Disclosure are
compounds represented by Formula (Ia):
##STR00068##
[0127] wherein
[0128] R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are hydrogen;
[0129] X.sup.1 and X.sup.2 are independently O, S or NR.sup.c;
wherein R.sup.c is hydrogen or C.sub.1-C.sub.4 alkyl,
[0130] Y is O, S or NR.sup.c; and
[0131] LG is a leaving group.
[0132] In another embodiment, Compounds of the Disclosure are
compounds represented by Formula (Ia) above; R.sup.1, R.sup.2,
R.sup.3, and R.sup.4 are hydrogen; X.sup.1 and X.sup.2 are S; Y is
S; and LG is represented by Formula (LG1) below:
##STR00069##
[0133] wherein
[0134] X.sup.3 is --O--, or --S--;
[0135] R* is independently hydrogen, deuterium, --CD.sub.3,
C.sub.1-C.sub.6 alkyl, --OH, halogen, --CN, --CF.sub.3. --NO.sub.2,
--O--C.sub.1-C.sub.6 alkyl, --O-aryl, --O-heteroaryl.
--O--C.sub.3-C.sub.8 cycloalkyl, --O-- heterocyclyl,
--NR.sup.bR.sup.b, --COOR.sup.b, or --CONR.sup.bR.sup.b, where
R.sup.a and R.sup.b are as defined above; and
[0136] t is 0, 1, 2, 3, 4, or 5.
[0137] In another embodiment, Compounds of the Disclosure are any
one or more of the compounds of Table 2.
TABLE-US-00002 TABLE 2 Cpd. No. Structure Name 2-1 ##STR00070##
2-(4-nitrophenoxy)-1,3,2-dithiaphospholane 2-sulfide 2-2
##STR00071## 2-phenoxy-1,3,2-dithiaphospholane 2-sulfide 2-3
##STR00072## 2-(phenylthio)-1,3,2-dithiaphospholane 2-sulfide 2-4
##STR00073## 2-((4-nitrophenyl)thio)-1,3,2-dithiaphospholane
2-sulfide 2-5 ##STR00074##
2-(4-bromophenoxy)-1,3,2-dithiaphospholane 2-sulfide 2-6
##STR00075## 2-(4-chlorophenoxy)-1,3,2-dithiaphospholane 2-sulfide
2-7 ##STR00076## 2-((4-chlorophenyl)thio)-1,3,2-dithiaphospholane
2-sulfide 2-8 ##STR00077##
2-(perfluorophenoxy)-1,3,2-dithiaphospholane 2- sulfide 2-9
##STR00078## 2-((perfluorophenyl)thio)-1,3,2- dithiaphospholane
2-sulfide 2-10 ##STR00079##
2-(4-methoxyphenoxy)-1,3,2-dithiaphospholane 2-sulfide 2-11
##STR00080## 2-((4-methoxyphenyl)thio)-1,3,2- dithiaphospholane
2-sulfide 2-12 ##STR00081## 2-(4-(trifluoromethyl)phenoxy)-1,3,2-
dithiaphospholane 2-sulfide 2-13 ##STR00082##
4-((2-sulfido-1,3,2-dithiaphospholan-2- yl)oxy)benzonitrile 2-14
##STR00083## 2-(4-fluorophenoxy)-1,3,2-dithiaphospholane 2-sulfide
2-15 ##STR00084## 2-(3,5-difluorophenoxy)-1,3,2-dithiaphospholane
2-sulfide 2-16 ##STR00085##
2-(3,5-bis(trifluoromethyl)phenoxy)-1,3,2- dithiaphospholane
2-sulfide 2-17 ##STR00086## 2-(3,4,5-trifluorophenoxy)-1,3,2-
dithiaphospholane 2-sulfide 2-18 ##STR00087##
2-(2,4,6-tribromophenoxy)-1,3,2- dithiaphospholane 2-sulfide 2-19
##STR00088## 2-(perchlorophenoxy)-1,3,2-dithiaphospholane 2-sulfide
2-20 ##STR00089## 2-phenoxy-1,3,2-dithiaphosphinane 2-sulfide 2-21
##STR00090## 2-(4-bromophenoxy)-1,3,2-dithiaphosphinane
2-sulfide
[0138] In one embodiment, Compounds of the Disclosure is compound
2-1. In another embodiment, Compounds of the Disclosure include one
or more compounds selected from the group consisting of compounds
2-2 to 2-21.
[0139] The present disclosure further includes all possible
stereoisomers and geometric isomers of Compounds of the Disclosure
to include both racemic compounds and optically active isomers.
When a Compound of the Disclosure is desired as a single
enantiomer, it can be obtained either by resolution of the final
product or by stereospecific synthesis from either isomerically
pure starting material or use of a chiral auxiliary reagent, for
example, see Z. Ma et al., Tetrahedron: Asymmetry, 8(6), pages
883-888 (1997). Resolution of the final product, an intermediate,
or a starting material can be achieved by any suitable method known
in the art. Additionally, in situations where tautomers of the
Compounds of the Disclosure are possible, the present disclosure is
intended to include all tautomeric forms of the compounds.
b. Nucleoside-Loaded Reagents
[0140] In another aspect, the present disclosure provides compounds
represented by any one of Formulae (IV)-(VIe). In one embodiment,
the compounds of the present disclosure are represented by Formula
(IV):
##STR00091##
[0141] wherein
[0142] (a) R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6
are independently hydrogen, CD.sub.3 or CF.sub.3, linear or
branched C.sub.1-C.sub.20 alkyl, optionally substituted with one or
more, the same or different. R.sup.a groups; linear or branched
C.sub.2-C.sub.12 alkenyl; optionally substituted with one or more,
the same or different, R.sup.a groups; linear or branched
C.sub.2-C.sub.12 alkynyl, optionally substituted with one or more,
the same or different, R.sup.a groups; aryl, optionally substituted
with one or more, the same or different, R.sup.a groups;
heteroaryl, optionally substituted with one or more, the same or
different, R.sup.a groups; heterocyclyl, optionally substituted
with one or more, the same or different, R.sup.a groups; or
C.sub.3-C.sub.8 cycloalkyl, optionally substituted with one or
more, the same or different, R.sup.a groups;
[0143] or
[0144] (b) any two of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R, and
R.sup.6 together with the carbons to which they are attached form a
C.sub.4-C.sub.8 cycloalkyl group, optionally substituted with one
or more, the same or different, R.sup.a groups, while the remaining
R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are as
defined in (a);
[0145] R.sup.a is hydrogen; deuterium, CD.sub.3, C.sub.1-C.sub.6
alkyl, OH, halogen, CN, CF.sub.3, O--C.sub.1-C.sub.6 alkyl, O-aryl,
O-heteroaryl, O--C.sub.3-C.sub.8 cycloalkyl, O-heterocyclyl,
--NR.sup.bR.sup.b, --COOR.sup.b or --CONR.sup.bR.sup.b;
[0146] R.sup.b is independently at each occurrence, the same or
different, hydrogen, linear or branched C.sub.1-C.sub.12 alkyl,
aryl, heteroaryl, heterocyclyl or C.sub.3-C.sub.8 cycloalkyl;
[0147] X.sup.1 and X.sup.2 are independently O, S or NR.sup.c;
wherein R.sup.c is hydrogen or C.sub.1-C.sub.4 alkyl;
[0148] Y is O, S or NR.sup.c;
[0149] m is 0, 1, or 2, and
[0150] Nu is a nucleoside;
[0151] provided that either the carbon bearing the R.sup.1 and
R.sup.2 groups, the carbon bearing the R.sup.3 and R.sup.4 groups,
or both, is chiral;
[0152] wherein the compound of formula (IV) is at least 90%
stereochemically pure,
[0153] with the proviso that Compound IV does not include any of
the following compounds:
##STR00092##
[0154] In one embodiment, the compound of the present disclosure
are represented by Formula (IV), wherein
[0155] (a) R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6
are independently hydrogen; linear or branched C.sub.1-C.sub.3
alkyl; C.sub.1-C.sub.3 alkyl substituted with halogen; CN; aryl;
heteroaryl; or C.sub.3-C.sub.6 cycloalkyl;
[0156] or
[0157] (b) any two of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
and R.sup.6 together with the carbons to which they are attached
form C.sub.4-C.sub.6 cycloalkyl, while the remaining R.sup.1,
R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are as defined in
(a);
[0158] provided that either the carbon bearing the R.sup.1 and
R.sup.2 groups, the carbon bearing the R.sup.3 and R.sup.4 groups,
or both, is chiral;
[0159] X.sup.1 and X.sup.2 are independently O, S or NR.sup.c,
wherein R.sup.c is hydrogen or C.sub.1-C.sub.4 alkyl;
[0160] Y is O, S or NR.sup.c;
[0161] m is independently 0, 1, or 2; and
[0162] Nu is a nucleoside.
[0163] In another embodiment, the compounds of the present
disclosure are represented by Formula (IVa):
##STR00093##
[0164] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, X.sup.1,
X.sup.2, Y, and Nu are as described for Formula (IV).
[0165] In another embodiment, the compounds of the present
disclosure are represented by Formulae (V-Vc):
##STR00094##
[0166] wherein
[0167] (a) R.sup.1, R.sup.3, R.sup.5 and R.sup.6 are independently
hydrogen, CD.sub.3 or CF.sub.3, linear or branched C.sub.1-C.sub.20
alkyl, optionally substituted with one or more, the same or
different, R.sup.a groups; linear or branched C.sub.2-C.sub.12
alkenyl; optionally substituted with one or more, the same or
different, R.sup.a groups; linear or branched C.sub.2-C.sub.12
alkynyl, optionally substituted with one or more, the same or
different, R.sup.a groups; aryl, optionally substituted with one or
more, the same or different, R.sup.a groups; heteroaryl, optionally
substituted with one or more, the same or different, R.sup.a
groups; heterocyclyl, optionally substituted with one or more, the
same or different, R.sup.a groups; or C.sub.3-C.sub.8 cycloalkyl,
optionally substituted with one or more, the same or different,
R.sup.a groups; or
[0168] (b) any two of R.sup.1, R.sup.3, R.sup.5, and R.sup.6
together with the carbons to which they are attached form a
C.sub.4-C.sub.8 cycloalkyl group, optionally substituted with one
or more, the same or different, R.sup.a groups while the remaining
R.sup.1, R.sup.3, R.sup.5, and R.sup.6 are as defined in (a);
[0169] R.sup.a is hydrogen; deuterium, CD.sub.3, C.sub.1-C.sub.6
alkyl, OH, halogen, CN, CF.sub.3, O--C.sub.1-C.sub.6 alkyl, O-aryl,
O-heteroaryl, O--C.sub.3-C.sub.8 cycloalkyl, O-heterocyclyl,
--NR.sup.bR.sup.b, --COOR.sup.b or --CONR.sup.bR.sup.b;
[0170] R.sup.b is independently at each occurrence, the same or
different, hydrogen, linear or branched C.sub.1-C.sub.12 alkyl,
aryl, heteroaryl, heterocyclyl or C.sub.3-C.sub.8 cycloalkyl;
[0171] m is 0, 1, or 2;
[0172] Y is O, S or NR.sup.c, wherein R.sup.c is hydrogen or
C.sub.1-C.sub.4 alkyl; and
[0173] Nu is a nucleoside.
[0174] In one embodiment, the compounds of the present disclosure
are represented by Formula (V), wherein
[0175] (a) R.sup.1, R.sup.3, R.sup.5 and R.sup.6 are independently
hydrogen; linear or branched C.sub.1-C.sub.3 alkyl; C.sub.1-C.sub.3
alkyl substituted with halogen; CN; aryl; heteroaryl; or
C.sub.3-C.sub.6 cycloalkyl;
[0176] or
[0177] (b) any two of R.sup.1, R.sup.3, R.sup.5, and R.sup.6
together with the carbons to which they are attached form
C.sub.4-C.sub.6 cycloalkyl, while the remaining R.sup.1, R.sup.3,
R.sup.5, and R.sup.6 are as defined in (a);
[0178] provided that either the carbon bearing the R.sup.1 and
R.sup.2 groups, the carbon bearing the R.sup.3 and R.sup.4 groups,
or both, is chiral;
[0179] Y is O, S or NR.sup.c, wherein R.sup.c is hydrogen or
C.sub.1-C.sub.4 alkyl;
[0180] m is 0, 1, or 2, and
[0181] Nu is a nucleoside.
[0182] In another embodiment, the compounds of the present
disclosure are represented by any one of Formulae (Vd-Vg):
##STR00095##
[0183] wherein R.sup.1, R.sup.3 and Nu are as described for
Formulae (V-Vc).
[0184] In yet another embodiment, the compounds of the present
disclosure are represented by any one of Formulae (VI-VIc):
##STR00096##
[0185] wherein
[0186] R.sup.2 and R.sup.4 are independently hydrogen; linear or
branched C.sub.1-C.sub.6 alkyl, optionally substituted with one or
more, the same or different, R.sup.a groups; linear or branched
C.sub.2-C.sub.6 alkenyl, optionally substituted with one or more,
the same or different, R.sup.a groups; linear or branched
C.sub.2-C.sub.6 alkynyl, optionally substituted with one or more,
the same or different, R.sup.a groups; halogen, --CN, --NO.sub.2;
wherein
[0187] R.sup.a is hydrogen; deuterium, CD.sub.3, C.sub.1-C.sub.6
alkyl, OH, halogen, CN, CF.sub.3, O--C.sub.1-C.sub.6 alkyl, O-aryl,
O-heteroaryl, O--C.sub.3-C.sub.8 cycloalkyl, O-heterocyclyl,
--NR.sup.bR.sup.b, --COOR.sup.b or --CONR.sup.bR.sup.b;
[0188] R.sup.b is independently at each occurrence, the same or
different, hydrogen, linear or branched C.sub.1-C.sub.12 alkyl,
aryl, heteroaryl, heterocyclyl or C.sub.3-C.sub.8 cycloalkyl;
and
[0189] Nu is a nucleoside.
[0190] In yet another embodiment, the compounds of the present
disclosure are represented by Formula (VId)
##STR00097##
[0191] wherein
[0192] R.sup.2 and R.sup.4 are independently hydrogen, CD.sub.3 or
CF.sub.3, linear or branched C.sub.1-C.sub.20 alkyl, optionally
substituted with one or more, the same or different, R.sup.a
groups; linear or branched C.sub.2-C.sub.12 alkenyl; optionally
substituted with one or more, the same or different, R.sup.a
groups; linear or branched C.sub.2-C.sub.12 alkynyl, optionally
substituted with one or more, the same or different, R.sup.a
groups; aryl, optionally substituted with one or more, the same or
different. R.sup.a groups; heteroaryl, optionally substituted with
one or more, the same or different, R.sup.a groups; heterocyclyl,
optionally substituted with one or more, the same or different,
R.sup.a groups; or C.sub.3-C.sub.8 cycloalkyl, optionally
substituted with one or more, the same or different, R.sup.a
groups;
[0193] R.sup.a is hydrogen; deuterium, CD.sub.3, C.sub.1-C.sub.6
alkyl, OH, halogen, CN, CF.sub.3, O--C.sub.1-C.sub.6 alkyl, O-aryl,
O-heteroaryl, O--C.sub.3-C.sub.8 cycloalkyl, O-heterocyclyl,
--NR.sup.bR.sup.b, --COOR.sup.b or --CONR.sup.bR.sup.b;
[0194] R.sup.b is independently at each occurrence, the same or
different, hydrogen, linear or branched C.sub.1-C.sub.12 alkyl,
aryl, heteroaryl, heterocyclyl or C.sub.3-C.sub.8 cycloalkyl;
[0195] R.sup.7 is hydrogen; CD.sub.3, OH, halogen, CN, CF.sub.3,
linear or branched C.sub.1-C.sub.6 alkyl; linear or branched
C.sub.2-C.sub.6 alkenyl, or linear or branched C.sub.2-C.sub.6
alkynyl;
[0196] X.sup.1 and X.sup.2 are independently O, S or NR.sup.c,
wherein R.sup.c is hydrogen or C.sub.1-C.sub.4 alkyl;
[0197] Y is O, S or NR.sup.c;
[0198] n is 0, 1, 2, 3 or 4; and
[0199] Nu is a nucleoside.
[0200] In yet another embodiment, the compounds of the present
disclosure are represented by any one of Formula (Vie):
##STR00098##
[0201] wherein
[0202] R.sup.2 and R.sup.4 are independently hydrogen, CD.sub.3 or
CF.sub.3, linear or branched C.sub.1-C.sub.20 alkyl, optionally
substituted with one or more, the same or different, R.sup.a
groups; linear or branched C.sub.2-C.sub.12 alkenyl; optionally
substituted with one or more, the same or different, R.sup.a
groups; linear or branched C.sub.2-C.sub.12 alkynyl, optionally
substituted with one or more, the same or different, R.sup.a
groups; aryl, optionally substituted with one or more, the same or
different, R.sup.a groups; heteroaryl, optionally substituted with
one or more, the same or different, R.sup.a groups; heterocyclyl,
optionally substituted with one or more, the same or different,
R.sup.a groups; or C.sub.3-C.sub.8 cycloalkyl, optionally
substituted with one or more, the same or different, R.sup.a
groups;
[0203] R.sup.a is hydrogen; deuterium, CD.sub.3, C.sub.1-C.sub.6
alkyl, OH, halogen, CN, CF.sub.3, O--C.sub.1-C.sub.6 alkyl, O-aryl,
O-heteroaryl, O--C.sub.3-C.sub.8 cycloalkyl, O-heterocyclyl,
--NR.sup.bR.sup.b, --COOR.sup.b or --CONR.sup.bR.sup.b;
[0204] R.sup.b is independently at each occurrence, the same or
different, hydrogen, linear or branched C.sub.1-C.sub.12 alkyl,
aryl, heteroaryl, heterocyclyl or C.sub.3-C.sub.8 cycloalkyl;
[0205] provided that either the carbon bearing the R.sup.1 or
R.sup.2 groups, the carbon bearing the
[0206] R.sup.3 or R.sup.4 groups, or both, is chiral;
[0207] X.sup.1 and X.sup.2 are independently O, S or NR.sup.c,
wherein R.sup.c is hydrogen or C.sub.1-C.sub.4 alkyl;
[0208] Y is O, S or NR.sup.c; and
[0209] Nu is a nucleoside.
[0210] In one embodiment, the nucleoside is a ribonucleoside. In
another embodiment, the nucleoside is deoxyribonucleoside.
[0211] Nucleosides can be naturally occurring nucleosides, or
non-naturally occurring nucleoside analogs. "Nucleoside analogs" as
used herein are variants of natural nucleosides, such as DNA or RNA
nucleosides, by virtue of modifications in the sugar and/or base
moieties. Analogs could in principle be merely "silent" or
"equivalent" to the natural nucleosides in the context of an
oligonucleotide, i.e. have no functional effect on the way the
oligonucleotide works. Such "equivalent" analogs can nevertheless
be useful if, for example, they are easier or cheaper to
manufacture, or are more stable to storage or manufacturing
conditions, or represent a tag or label. In some embodiments,
however, the analogs will have a functional effect on the way in
which the oligonucleotide functions; for example by producing
increased binding affinity to the target and/or increased
resistance to intracellular nucleases and/or increased ease of
transport into the cell.
[0212] Useful nucleosides employed herein can also include modified
sugars, 2'-sugar modifications include fluoro, O-alkyl,
O-alkylamino, O-alkylalkoxy, protected O-alkylamino,
O-alkylaminoalkyl, O-alkyl imidazole, and polyethers of the formula
(O-alkyl).sub.m, where m is 1 to about 10. Preferred among these
polyethers are linear and cyclic polyethylene glycols (PEGs), and
PEG-containing groups, such as crown ethers and those which are
disclosed by Ouchi et al., Drug Design and Discovery 1992, 9, 93;
Ravasio et al., J. Org. Chem. 1991, 56, 4329; and Delgardo et. al.,
Critical Reviews in Therapeutic Drug Carrier Systems 1992, 9, 249,
each of which is hereby incorporated by reference in its entirety.
Further nucleosides embodying sugar modifications are disclosed in
Cook. Anti-Cancer Drug Design, 1991, 6, 585-607 and US Publication
No. 2016/237427, hereby incorporated by reference in their
entirety. Fluoro, O-alkyl, O-alkylamino, O-alkyl imidazole,
O-alkylaminoalkyl, and alkyl amino substitutions are described in
U.S. Pat. No. 6,166,197, entitled "Oligomeric Compounds having
Pyrimidine Nucleotide(s) with 2' and 5' Substitutions," hereby
incorporated by reference in its entirety.
[0213] Additional useful nucleosides having 2'-sugar modifications
include 2'-SR and 2'-NR.sub.2 groups, where each R is,
independently, hydrogen, a protecting group or substituted or
unsubstituted alkyl, alkenyl, or alkynyl, 2'-SR nucleosides are
disclosed in U.S. Pat. No. 5,670,633, issued Sep. 23, 1997, hereby
incorporated by reference in its entirety.
[0214] Useful nucleosides also include nucleosides derivatized with
selenium (Se). Examples of Se-derivatized nucleosides include
nucleosides where O-atom at the positions 2', and/or 5' of the
sugar have been replaced with Se. Other examples include oxygen
replacement with Se in the furanose ring, nucleobases and
non-bridging phosphates. Such nucleic acids are described in, for
example, Pallan et al., Nat. Protoc., 2(3):647-51 (2007), and Nat.
Protoc., 2(3); 640-646 (2007), hereby incorporated by reference in
their entirety.
[0215] Other examples of suitable nucleosides include boron
containing nucleosides, such as those described in Schinazi et al.,
Nucleosides and Nucleotides, 17(635-647 (1998); Biochem.,
35(18):5741-5746 (1996); J. Org. Chem., 79(8):3465-3472 (2014),
hereby incorporated by reference in their entirety.
[0216] Additional useful nucleoside analogs include, but are not
limited to, Locked Nucleic Acid (LNA); 2'-O-alkyl-RNA;
2'-amino-DNA; 2'-fluoro-DNA; arabino nucleic acid (ANA);
2'-fluoro-ANA, hexitol nucleic acid (HNA), intercalating nucleic
acid (INA), constrained ethyl nucleoside (cEt), 2'-O-methyl nucleic
acid (2'-OMe), 2'-O-methoxyethyl nucleic acid (2'-MOE), or any
combination thereof.
[0217] "Hexitol nucleic acids" or "HNA" are composed of
phosphorylated 2,3-dideoxy-D-arabino-hexitol units with a
nucleobase situated in the 2-[S]-position.
[0218] "cEt" or "constrained ethyl" means a bicyclic nucleoside
having a sugar moiety comprising a bridge connecting the 4'-carbon
and the 2'-carbon, wherein the bridge has the formula:
4'-CH(CH.sub.3)-0-2'.
[0219] "2'-O-methoxyethyl" (also 2'-MOE and
2'-O(CH.sub.2).sub.2--OCH.sub.3 and MOE) refers to an
O-methoxy-ethyl modification at the 2' position of a furanosyl
ring. A 2'-O-methoxyethyl modified sugar is a modified sugar.
[0220] "2'-F" refers to modification of the 2' position of the
furanosyl sugar ring to comprise a fluoro group.
[0221] As used herein, "2'-OMe" or "2'-OCH.sub.3" or "2'-O-methyl"
each refers to modification at the 2' position of the furanosyl
sugar ring to comprise a --OCH.sub.3 group.
[0222] Examples of suitable nucleotide analogs are provided by
WO2007/031091, which is incorporated by reference in its entirety,
or are referenced therein.
[0223] Incorporation of affinity-enhancing nucleotide analogs in an
oligomer, such as LNA or 2'-substituted sugars, can allow the size
of the specifically binding oligomer to be reduced, and can also
reduce the upper limit to the size of the oligomer before
non-specific or aberrant binding takes place.
[0224] In some embodiments, the nucleoside is a nucleoside analog
that includes a bicyclic sugar. Non-limiting examples of the
bicyclic sugar includes cEt, 2',4'-constrained 2'-O-methoxyethyl
(cMOE), LNA, .alpha.-LNA, .beta.-LNA, 2'-O,4'-C-ethylene-bridged
nucleic acids (ENA), amino-LNA, oxy-LNA, or thio-LNA.
[0225] The term "LNA" refers to a bicyclic nucleoside analog, known
as "Locked Nucleic Acid". It can refer to an LNA monomer, or, when
used in the context of an "LNA oligonucleotide," LNA refers to an
oligonucleotide containing one or more such bicyclic nucleotide
analogs. LNA nucleosides are characterized by the presence of a
linker group (such as a bridge) between C2' and C4' of the ribose
sugar ring. This bridge includes, but is not limited to, a
biradical (bivalent group) selected from --CH.sub.2--O--,
--CH.sub.2--S--, --CH.sub.2--NH--, --CH.sub.2--N(CH.sub.3)--,
--CH.sub.2--CH.sub.2--O--, --CH.sub.2--CH(CH.sub.3)--,
--CH.sub.2--CH.sub.2--S--, --CH.sub.2--CH.sub.2--NH--,
--CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--CH.sub.2--O--,
--CH.sub.2--CH.sub.2--CH(CH.sub.3)--, --CH--CH--CH.sub.2--,
--CH.sub.2--O--CH.sub.2--O--, --CH.sub.2--NH--O--,
--CH.sub.2--N(CH.sub.3)--O--, --CH.sub.2--O--CH.sub.2--,
--CH(CH.sub.3)--O--, and --CH(CH.sub.2--O--CH.sub.3)--O--, and/or,
--CH.sub.2--CH.sub.2--, and --CH.dbd.CH-- For all chiral centers,
asymmetric groups can be found in either R or S orientation.
[0226] In some embodiments, the biradical can be
--C(R.sup.aR.sup.b)--O--C(R.sup.cR.sup.d)--O--, wherein R.sup.a,
R.sup.b, R.sup.c, and R.sup.d are independently selected from the
group consisting of hydrogen, halogen, C.sub.1-6 alkyl, substituted
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, substituted C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl or substituted C.sub.2-6 alkynyl, C.sub.1-6
alkoxyl, substituted C.sub.1-6 alkoxyl, acyl, substituted acyl,
C.sub.1-6 aminoalkyl or substituted C.sub.1-6 aminoalkyl, such as
hydrogen.
[0227] Suitable bicyclic nucleosides are disclosed in WO
2007/134181, WO2008/154401, WO2008/150729. WO2009/067647
(alpha-L-bicyclic nucleic acids analogs) and WO2009006478A, all of
which are hereby incorporated by reference in its entirety.
[0228] Further hicyclic nucleoside analogs and their use in
antisense oligonucleotides are disclosed in WO2011/115818,
WO2011/085102, WO2011/017521, WO2009/100320, WO2010/036698,
WO2009/124295 and WO2009/006478, each of which are incorporated by
reference herein in their entireties.
[0229] The term "thio-LNA" comprises a locked nucleoside in which Y
in general Formula III below is selected from S or --CH.sub.2--S--.
Thio-LNA can be in both beta-D and alpha-L-configuration.
[0230] The term "amino-LNA" comprises a locked nucleoside in which
Y in general Formula III below is selected from --N(H)--, N(R)--,
CH.sub.2--N(H)--, and --CH.sub.2--N(R)-- where R is selected from
hydrogen and C.sub.1-4-alkyl. Amino-LNA can be in both beta-D and
alpha-L-configuration.
[0231] The term "oxy-LNA" comprises a locked nucleoside in which Y
in general Formula III below represents --O--. Oxy-LNA can be in
both beta-D and alpha-L-configuration.
[0232] The term "ENA" comprises a locked nucleoside in which Y in
general Formula III below is --CH.sub.2--O-- (where the oxygen atom
of --CH.sub.2--O-- is attached to the 2'-position relative to the
base B). R.sup.e is hydrogen or methyl.
[0233] In some exemplary embodiments LNA is selected from
beta-D-oxy-LNA, alpha-L-oxy-LNA, beta-D-amino-LNA and
beta-D-thio-LNA.
[0234] In some embodiments the nucleoside analogs can be, for
example: 2'-O-alkyl-RNA units, 2'-amino-DNA units, 2'-fluoro-DNA
units, LNA units, arabino nucleic acid (ANA) units, 2'-fluoro-ANA
units. HNA units. INA (intercalating nucleic acid--Christensen,
2002. Nucl. Acids. Res. 2002 30: 4918-4925, hereby incorporated by
reference) units and 2'-MOE units.
[0235] The term "nucleobase" refers to the base moiety of a
nucleotide and covers both naturally occurring as well as
non-naturally occurring variants. Thus, "nucleobase" covers not
only the known purine and pyrimidine heterocycles but also
heterocyclic analogs and tautomers thereof.
[0236] Typical nucleobases include, but are not limited to adenine,
guanine, cytosine, thymidine, uracil, xanthine, hypoxanthine,
5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil,
5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine,
diaminopurine, and 2-chloro-6-aminopurine.
[0237] In some embodiments, the nucleoside comprises a
naturally-occurring nucleobase, such as adenine, guanine, cytosine,
uridine, thymine, 5-methyl cytosine, etc. In other embodiments, the
nucleoside comprises other natural nucleobases, as well as modified
nucleobases, such as xanthine, hypoxanthine, 2-aminoadenine,
6-methyl and other alkyl derivatives of adenine and guanine,
2-propyl and other alkyl derivatives of adenine and guanine, 5-halo
uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil
(pseudo uracil), 4-thiouracil, 8-halo, oxa, amino, thiol,
thioalkyl, hydroxyl and other 8-substituted adenines and guanines,
5-trifluoromethyl and other 5-substituted uracils and cytosines,
7-methylguanine. Further naturally- and non-naturally-occurring
nucleobases include those disclosed in U.S. Pat. No. 3,687,808
(Merigan et al.); in Sanghvi, in Antisense Research and
Application, Chapter 15, S. T. Crooke and B. Lebleu, Eds., CRC
Press, 1993; in Englisch et al., Angewandte Chemie, International
Edition, 1991, 30, 613-722 (particularly, pages 622 and 623); in
the Concise Encyclopedia of Polymer Science and Engineering, J. I.
Kroschwitz, Ed., John Wiley & Sons, 1990, pages 858-859; in
Zhang, et al., Nature, 2017, 551, 644-647 (hydrophobic bases); in
Feldman and Romesberg, Acc. Chem. Res. 2018, 51, 394-403; and in
Cook, Anti-Cancer Drug Design, 1991, 6, 585-607, each of which is
hereby incorporated by reference in its entirety.
[0238] Other examples of modifications of nucleosides and
nucleobases described herein include, but are not limited to the
following: 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine;
2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonyl
carbamoyladenosine; N6-glycinylcarbamoyladenosine;
N6-isopentenyladenosine; N6-methyladenosine;
N6-threonylcarbamoyladenosine; 1,2'-O-dimethyladenosine;
1-methyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine
(phosphate); 2-methyladenosine; 2-methylthio-N6
isopentenyladenosine; 2-methylthio-N6-hydroxynorvalyl
carbamoyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine
(phosphate); Isopentenyladenosine;
N6-(cis-hydroxyisopentenyl)adenosine; N6,2'-O-dimethyladenosine;
N6,2'-O-dimethyladenosine; N6,N6,2'-O-triinethyladenosine;
N6,N6-dimethyladenosine; N6-acetyladenosine;
N6-hydroxynorvalylcarbamoyladenosine;
N6-methyl-N6-threonylcarbamoyladenosine;
2-methylthio-N6-isopentenyladenosine; 7-deaza-adenosine;
N1-methyl-adenosine; N6. N6 (dimethyl)adenine;
N6-cis-hydroxy-isopentenyl-adenosine; .alpha.-thio-adenosine; 2
(amino)adenine; 2 (aminopropyl)adenine; 2 (methylthio) N6
(isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adeniine;
2-(aminiopropyl)adenine; 2-(halo)adenine; 2-(propyl)adeniine;
2'-amino-2'-deoxy-adeniosine triphosphate;
2'-azido-2'-deoxy-adenosine triphosphate;
2'-deoxy-2'-.alpha.-aminoadenosine triphosphate;
2'-deoxy-2'-a-azidoadenosine triphosphate; 6-(alkyl)adenine;
6-(methyl)adenine; 7 (deaza)adenine; 8 (alkynyl)adenine;
8-(alkenyl)adenine; 8-(alkyl)adenine; 8-(alkynyl)adenine;
8-(amino)adenine; 8-(halo)adenine; 8-(hydroxyl)adenine;
8-(thioalkyl)adenine; 8-(thiol)adenine; 8-azido-adenosine; aza
adenine; deaza adenine; N6 (methyl)adenine; N6-(isopentyl)adenine;
7-deaza-8-aza-adenosine; 7-methyladenine; 1-deazaadenosine
triphosphate; 2'fluoro-N6-Bz-deoxyadenosine triphosphate;
2'-methoxy-2-amino-adenosine triphosphate;
2'O-methyl-N6-Bz-deoxyadenosine triphosphate; 2'-a-Ethynyladenosine
triphosphate; 2-aminoadenine; 2-aminoadenosine triphosphate;
2-amino-adenosine triphosphate; 2'-a-trifluoromethyladenosine
triphosphate; 2-azidoadenosine triphosphate; 2'-b-ethynyladenosine
triphosphate; 2-bromoadenosine triphosphate;
2'-b-trifluoromethyladenosine triphosphate; 2-chloroadenosine
triphosphate; 2'-deoxy-2',2'-difluoroadenosine triphosphate;
2'-deoxy-2'-a-mercaptoadenosine triphosphate;
2'-deoxy-2'-.alpha.-thiomethoxyadenosine triphosphate;
2'-deoxy-2'-b-aminoadenosine triphosphate;
2'-deoxy-2'-b-azidoadenosine triphosphate;
2'-deoxy-2'-b-bromoadenosine triphosphate;
2'-deoxy-2'-b-chloroadenosine triphosphate;
2'-deoxy-2'-b-fluoroadenosine triphosphate;
2'-deoxy-2'-b-iodoadenosine triphosphate;
2'-deoxy-2'-b-mercaptoadenosine triphosphate;
2'-deoxy-2'-b-thiomethoxyadenosine triphosphate; 2-fluoroadenosine
triphosphate; 2-iodoadenosine triphosphate; 2-mercaptoadenosine
triphosphate; 2-methoxy-adenine; 2-methylthio-adenine;
2-trifluoromethyladenosine triphosphate; 3-deaza-3-bromoadenosine
triphosphate; 3-deaza-3-chloroadenosine triphosphate;
3-deaza-3-fluoroadenosine triphosphate; 3-deaza-3-iodoadenosine
triphosphate; 3-deazaadenosine triphosphate; 4'-azidoadenosine
triphosphate; 4'-carbocyclic adenosine triphosphate;
4'-ethynyladenosine triphosphate; 5'-homo-adenosine triphosphate;
8-aza-adenosine triphosphate; 8-bromo-adenosine triphosphate;
8-Trifluoromethyladenosine triphosphate; 9-deazaadenosine
triphosphate; 2-aminopurine; 7-deaza-2,6-diaminopurine;
7-deaza-8-aza-2,6-diaminopurine; 7-deaza-8-aza-2-aminopurine:
2,6-diaminopurine; 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine;
2-thiocytidine; 3-methylcytidine; 5-formylcytidine;
5-hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine;
2'-O-methylcytidine; 5,2'-O-dimethylcytidine;
5-formyl-2'-O-methylcytidine; Lysidine; N4,2'-O-dimethylcytidine;
N4-acetyl-2'-O-methylcytidine; N4-methylcytidine;
N4,N4-Dimethyl-2'-OMe-Cytidine TP; 4-methylcytidine;
5-aza-cytidine; pseudo-iso-cytidine; pyrrolo-cytidine;
.alpha.-thio-cytidine; 2-(thio)cytosine; 2'-amino-2'-deoxy-cytidine
triphosphate; 2'-azido-2'-deoxy-cytidine triphosphate;
2'-deoxy-2'-a-aminocytidine triphosphate;
2'-deoxy-2'-a-azidocytidine triphosphate; 3 (deaza) 5
(aza)cytosine; 3 (methyl)cytosine; 3-(alkyl)cytosine; 3-(deaza) 5
(aza)cytosine; 3-(methyl)cytidine; 4,2'-O-dimethylcytidine; 5
(halo)cytosine; 5 (methyl)cytosine; 5 (propynyl)cytosine; 5
(trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine;
5-(halo)cytosine; 5-(propynyl)cytosine;
5-(trifluoromethyl)cytosine; 5-bromo-cytidine; 5-iodo-cytidine;
5-propynyl cytosine; 6-(azo)cytosine; 6-aza-cytidine; aza cytosine;
deaza cytosine; N4 (acetyl)cytosine;
1-methyl-1-deaza-pseudoisocytidine; 1-methyl-pseudoisocytidine;
2-methoxy-5-methyl-cytidine; 2-methoxy-cytidine;
2-thio-5-methyl-cytidine; 4-methoxy-1-methyl-pseudoisocytidine;
4-methoxy-pseudoisocytidine;
4-thio-1-methyl-1-deaza-pseudoisocytidine;
4-thio-1-methyl-pseudoisocytidine; 4-thio-pseudoisocytidine;
5-aza-zebularine; 5-methyl-zebularine; pyrrolo-pseudoisocytidine;
zebularine; (E)-5-(2-bromo-vinyl)cytidine triphosphate;
2,2'-anhydro-cytidine triphosphate hydrochloride;
2'fluor-N4-Bz-cytidine triphosphate; 2'fluoro-N4-acetyl-cytidine
triphosphate; 2'-O-methyl-N4-acetyl-cytidine triphosphate;
2'O-methyl-N4-Bz-cytidine triphosphate; 2'-a-ethynylcytidine
triphosphate; 2'-a-trifluoromethylcytidine triphosphate;
2'-b-ethynylcytidine triphosphate; 2'-b-trifluoromethylcytidine
triphosphate; 2'-deoxy-2',2'-difluorocytidine triphosphate;
2'-deoxy-2'-a-mercaptocytidine triphosphate;
2'-deoxy-2'-a-thiomethoxycytidine triphosphate;
2'-deoxy-2'-b-aminocytidine triphosphate;
2'-deoxy-2'-b-azidocytidine triphosphate;
2'-deoxy-2'-b-bromocytidine triphosphate;
2'-deoxy-2'-b-chlorocytidine triphosphate;
2'-deoxy-2'-b-fluorocytidine triphosphate;
2'-deoxy-2'-b-iodocytidine triphosphate;
2'-deoxy-2'-b-mercaptocytidine triphosphate;
2'-deoxy-2'-b-thiomethoxycytidine triphosphate;
2'-O-methyl-5-(1-propynyl)cytidine triphosphate; 3'-ethynylcytidine
triphosphate; 4'-azidocytidine triphosphate; 4'-carbocyclic
cytidine triphosphate; 4'-ethynylcytidine triphosphate;
5-(1-propynyl)ara-cytidine triphosphate;
5-(2-chloro-phenyl)-2-thiocytidine triphosphate;
5-(4-amino-phenyl)-2-thiocytidine triphosphate;
5-aminoallyl-cytidine triphosphate; 5-cyanocytidine triphosphate;
5-ethynylara-cytidine triphosphate; 5-ethynylcytidine triphosphate;
5'-homo-cytidine triphosphate; 5-methoxycytidine triphosphate;
5-trifluoromethyl-cytidine triphosphate; N4-amino-cytidine
triphosphate; N4-benzoyl-cytidine triphosphate; pseudoisocytidine;
7-methylguanosine; N2,2'-O-dimethylguanosine; N2-methylguanosine;
wyosine; 1,2'-O-dimethylguanosine; 1-methylguanosine;
2'-O-methylguanosine; 2'-O-ribosylguanosine (phosphate);
2'-O-methylguanosine; 2'-O-ribosylguanosine (phosphate);
7-aminomethyl-7-deazaguanosine; 7-cyano-7-deazaguanosine;
archaeosine; methylwyosine; N2,7-dimethylguanosine;
N2,N2,2'-O-trimethylguanosine; N2,N2,7-timethylguanosine;
N2,N2-dimethylguanosine; N2,7,2'-O-trimethylguanosine;
6-thio-guanosine; 7-deaza-guanosine; 8-oxo-guanosine;
N1-methyl-guanosine; .alpha.-thio-guanosine; 2 (propyl)guanine;
2-(alkyl)guanine; 2'-Amino-2'-deoxy-guanosine triphosphate;
2'-Azido-2'-deoxy-guanosine triphosphate;
2'-deoxy-2'-a-aminoguanosine triphosphate;
2'-deoxy-2'-a-azidoguanosine triphosphate; 6-(alkyl)guanine;
6-methyl-guanosine; 7-(alkyl)guanine; 7-(deaza)guanine;
7-(methyl)guanine; 8-(alkenyl)guanine; 8-(alkyl)guanine;
8-(alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine;
8-(hydroxyl)guanine; 8-(thioalkyl)guanine; 8-(thiol)guanine; aza
guanine; deaza guanine; N-(methyl)guanine;
1-methyl-6-thio-guanosine; 6-methoxy-guanosine;
6-thio-7-deaza-8-aza-guanosine; 6-thio-7-deaza-guanosine;
6-thio-7-methyl-guanosine; 7-deaza-8-aza-guanosine;
7-methyl-8-oxo-guanosine; N2,N2-dimethyl-6-thio-guanosine;
N2-methyl-6-thio-guanosine; 1-me-guanosine triphosphate;
2'fluoro-N2-isobutyl-guanosine triphosphate;
2'O-methyl-N2-isobutyl-guanosine triphosphate;
2'-a-ethynylguanosine triphosphate; 2'-a-trifluoromethylguanosine
triphosphate; 2'-b-ethynylguanosine triphosphate;
2'-b-trifluoromethylguanosine triphosphate;
2'-deoxy-2',2'-difluoroguanosine triphosphate;
2'-deoxy-2'-a-mercaptoguanosine triphosphate;
2'-deoxy-2'-a-thiomethoxyguanosine triphosphate;
2'-deoxy-2'-b-aminoguanosine triphosphate;
2'-deoxy-2'-b-azidoguanosine triphosphate;
2'-deoxy-2'-b-bromoguanosine triphosphate;
2'-deoxy-2'-b-chloroguanosine triphosphate;
2'-deoxy-2'-b-fluoroguanosine triphosphate;
2'-deoxy-2'-b-iodoguanosine triphosphate;
2'-deoxy-2'-b-mercaptoguanosine triphosphate;
2'-deoxy-2'-b-thiomethoxyguanosine triphosphate; 4'-azidoguanosine
triphosphate; 4'-carbocyclic guanosine triphosphate;
4'-ethynylguanosine triphosphate; 5'-homo-guanosine triphosphate;
8-bromo-guanosine triphosphate; 9-deazaguanosine triphosphate;
N2-isobutyl-guanosine triphosphate; 1-methylinosine; inosine;
1,2'-O-dimethylinosine; 7-methylinosine; 2'-O-methylinosine;
epoxyqucuosine; galactosyl-queuosine; mannosylqueuosine; queuosine;
allyamino-thymidine; azathymlidine; deaza thymidinie;
deoxy-thymidine; 2-thiouridine; 3-methyluridine;
5-carboxymethyluridine; 5-hydroxyuridine; 5-methyluridine;
5-taurinomethyl-2-thiouridine; 5-taurinomethyluridine;
dihydrouridine; pseudouridine;
1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine;
1-methylpseduouridine; 1-ethyl-pseudouridine; 2'-O-methyluridine;
2'-O-methylpseudouridine; 2'-O-methyluridine;
2-thio-2'-O-methyluridine; 3-(3-amino-3-carboxypropyl)uridine;
3,2'-O-dimethyluridine; 3-methyl-pseudo-uridine triphosphate;
4-thiouridine; 5-(carboxyhydroxymethyl)uridine;
5-(carboxyhydroxymethyl)uridine methyl ester;
5,2'-O-dimethyluridine; 5,6-dihydro-uridine;
5-aminomethyl-2-thiouridine; 5-carbamoylmethyl-2'-O-methyluridine;
5-carbamoylmethyluridine; 5-carboxyhydroxymethyluridine;
5-carboxyhydroxymethyluridine methyl ester,
5-carboxymethylaminomethyl-2'-O-methyluridine;
5-carboxymethylaminomethyl-2-thiouridine;
5-carboxymethylaminomiethyluridine; 5-carhamoylmethyluridine
triphosphate; 5-methoxycarbonylmethyl-2'-O-methyluridine;
5-methoxycarbonylmethyl-2-thiouridine;
5-methoxycarbonylmethyluridine; 5-methoxyuridine;
5-methyl-2-thiouridine; 5-methylaminomethyl-2-selenouridine;
5-methylaminomethyl-2-thiouridine; 5-methylaminomethyluridine;
5-methyldihydrouridine; 5-oxyacetic acid-uridine triphosphate;
5-oxyacetic acid-methyl ester-uridine triphosphate;
N1-methyl-pseudo-uracil; N1-ethyl-pseudo-uracil; uridine
5-oxyacetic acid; uridine 5-oxyacetic acid methyl ester;
3-(3-Amino-3-carboxypropyl)-uridine triphosphate;
5-(iso-pentenylaminomethyl)-2-thiouridine triphosphate;
5-(iso-pentenylaminomethyl)-2'-O-methyluridine triphosphate;
5-(iso-pentenylaminomethyl)uridine triphosphate; 5-propynyl uracil;
.alpha.-thio-uridine; 1
(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil; 1
(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1
(aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouracil; 1
(aminoalkylaminocarbonylethylenyl)-pseudouracil; 1
(aminocarbonylethylenyl)-2(thio)-pseudouracil; 1 (amino
carbonylethylenyl)-2,4-(dithio)pseudouracil; 1
(aminocarbonylethylenyl)-4 (thio)pseudouracil; 1
(aminocarbonylethylenyl)-pseudouracil; 1 substituted
2(thio)-pseudouracil; 1 substituted 2,4-(dithio)pseudouracil; 1
substituted 4 (thio)pseudouracil; 1 substituted pseudouracil;
1-(aminoalkylamino-carbonylethylenyl)-2-(thio)-pseudouracil;
1-Methyl-3-(3-amino-3-carboxypropyl) pseudouridine triphosphate;
1-methyl-3-(3-amino-3-carboxypropyl)pseudo-uridine triphosphate;
I-methyl-pseudo-uridine triphosphate; 1-ethyl-pseudo-uridine
triphosphate; 2 (thio)pseudouracil; 2' deoxy uridine; 2'
fluorouridine; 2-(thio)uracil; 2,4-(dithio)psuedouracil; 2' methyl,
2'amino, 2'azido, 2'fluro-guanosine; 2'-amino-2'-deoxy-uridine
triphosphate; 2'-azido-2'-deoxy-uridine triphosphate;
2'-azido-deoxyuridine triphosphate; 2' deoxy uridine; 2'
fluorouridine; 2'-deoxy-2'-a-aminouridine triphosphate;
2'-deoxy-2'-a-azidouridine triphosphate; 2-methylpseudouridine; 3
(3 amino-3 carboxypropyl)uracil; 4-(thio)pseudouracil;
4-thiouracil; 5 (1,3-diazole-1-alkyl)uracil; 5
(2-aminopropyl)uracil; 5 (aminoalkyl)uracil; 5
(dimethylaminoalkyl)uracil; 5 (guanidiniumalkyl)uracil: 5
(methoxycarbonylmethyl)-2-(thio)uracil; 5
(methoxycarbonyl-methyl)uracil; 5 (methyl) 2 (thio)uracil; 5
(methyl) 2.4 (dithio)uracil; 5 (methyl) 4 (thio)uracil; 5
(methylaminomethyl)-2 (thio)uracil; 5 (methylaminomethyl)-2,4
(dithio)uracil; 5 (methylaminomethyl)-4 (thio)uracil; 5
(propynyl)uracil; 5 (trifluoromethyl)uracil;
5-(2-aminiopropyl)uracil; 5-(alkyl)-2-(thio)pseudouracil;
5-(alkyl)-2,4 (dithio)pseudouracil; 5-(alkyl)-4 (thio)pseudouracil;
5-(alkyl)pseudouracil; 5-(alkyl)uracil; 5-(alkynyl)uracil;
5-(allylamino)uracil; 5-(cyanoalkyl)uracil;
5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil;
5-(guanidiniumalkyl)uracil; 5-(halo)uracil;
5-(1,3-diazole-1-alkyl)uracil; 5-(methoxy)uracil;
5-(methoxycarbonylmethyl)-2-(thio)uracil;
5-(methoxycarbonyl-methyl)uracil; 5-(methyl) 2(thio)uracil;
5-(methyl) 2,4 (dithio)uracil; 5-(methyl) 4 (thio)uracil;
5-(methyl)-2-(thio)pseudouracil; 5-(methyl)-2,4
(dithio)pseudouracil; 5-(methyl)-4 (thio)pseudouracil;
5-(methyl)pseudouracil; 5-(methylaminomethyl)-2 (thio)uracil;
5-(methylaminomethyl)-2,4(dithio)uracil;
5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil;
5-(trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine;
5-iodo-uridine; 5-uracil; 6-(azo)uracil; 6-aza-uridine;
allyamino-uracil; aza uracil; deaza uracil; N3 (methyl)uracil;
pseudo-uridine triphosphate-1-2-ethanoic acid; pseudouracil;
4-thio-pseudo-uridine triphosphate; 1-carboxymethyl-pseudouridine;
1-methyl-1-deaza-pseudouridine; 1-propynyl-uridine;
1-taurinomethyl-1-methyl-uridine; 1-taurinomethyl-4-thio-uridine;
1-taurinomethyl-pseudouridine; 2-methoxy-4-thio-pseudouridine;
2-thio-1-methyl-1-deaza-pseudouridine;
2-thio-1-methyl-pseudouridine; 2-thio-5-aza-uridine;
2-thio-dihydropseudouridine; 2-thio-dihydrouridine;
2-thio-pseudouridine; 4-methoxy-2-thio-pseudouridine;
4-methoxy-pseudouridine; 4-thio-1-methyl-pseudouridine;
4-thio-pseudouridine; 5-aza-uridine; dihydropseudouridine; (.+-.)
1-(2-hydroxypropyl)pseudouridine triphosphate;
(2R)-1-(2-hydroxypropyl)pseudouridine triphosphate;
(2S)-1-(2-hydroxypropyl)pseudouridine triphosphate;
(E)-5-(2-bromo-vinyl)ara-uridine triphosphate;
(E)-5-(2-bromo-vinyl)uridine triphosphate;
(Z)-5-(2-Bromo-vinyl)ara-uridine triphosphate;
(Z)-5-(2-bromo-vinyl)uridine triphosphate;
1-(2,2,2-trifluoroethyl)-pseudo-uridine triphosphate;
1-(2,2,3,3,3-pentafluoropropyl)pseudouridine triphosphate;
1-(2,2-diethoxyethyl)pseudouridine triphosphate;
1-(2,4,6-trimethylbenzyl)pseudouridine triphosphate;
1-(2,4,6-trimethyl-benzyl)pseudo-uridine triphosphate;
1-(2,4,6-trimethyl-phenyl)pseudo-uridine triphosphate;
1-(2-amino-2-carboxyethyl)pseudo-uridine triphosphate;
1-(2-amino-ethyl)pseudo-uridine triphosphate;
1-(2-hydroxyethyl)pseudouridine triphosphate;
1-(2-methoxyethyl)pseudouridine triphosphate;
1-(3,4-bis-trifluoromethoxybenzyl)pseudouridine triphosphate;
1-(3,4-dimethoxybenzyl)pseudouridine triphosphate;
1-(3-amino-3-carboxypropyl)pseudo-uridine triphosphate;
1-(3-amino-propyl)pseudo-uridine triphosphate;
1-(3-cyclopropyl-prop-2-ynyl)pseudouridinetriphosphate;
1-(4-amino-4-carboxybutyl)pseudo-uridine triphosphate;
1-(4-amino-benzyl)pseudo-uridine triphosphate;
1-(4-amino-butyl)pseudo-uridine triphosphate;
1-(4-amino-phenyl)pseudo-uridine triphosphate;
1-(4-azidobenzyl)pseudouridine triphosphate;
1-(4-bromobenzyl)pseudouridine triphosphate;
1-(4-chlorobenzyl)pseudouridine triphosphate;
1-(4-fluorobenzyl)pseudouridine triphosphate;
1-(4-iodobenzyl)pseudouridine triphosphate;
1-(4-methanesulfonylbenzyl)pseudouridine triphosphate;
1-(4-methoxybenzyl)pseudouridine triphosphate;
1-(4-methoxy-benzyl)pseudo-uridine triphosphate;
1-(4-methoxy-phenyl)pseudo-uridine triphosphate;
1-(4-methyl-benzyl)pseudo-uridine triphosphate;
1-(4-nitro-benzyl)pseudo-uridine triphosphate; 1
(4-nitro-phenyl)pseudo-uridine triphosphate;
1-(4-thiomethoxybenzyl)pseudouridine triphosphate;
1-(4-trifluoromethoxybenzyl)pseudouridine triphosphate;
1-(4-trifluoromethylbenzyl)pseudouridine triphosphate;
1-(5-amino-pentyl)pseudo-uridine triphosphate;
1-(6-amino-hexyl)pseudo-uridine triphosphate;
1,6-dimethyl-pseudo-uridine triphosphate;
1-[3-(2-{2-[2-(2-aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouri-
dine triphosphate;
1-{3-[2-(2-aminoethoxy)-ethoxy]-propionyl}pseudouridine
triphosphate; 1-acetylpseudouridine triphosphate;
1-alkyl-6-(1-propynyl)-pseudo-uridine triphosphate;
1-alkyl-6-(2-propynyl)-pseudo-uridine triphosphate;
1-alkyl-6-allyl-pseudo-uridine triphosphate;
1-alkyl-6-ethynyl-pseudo-uridine triphosphate;
1-alkyl-6-homoallyl-pseudo-uridine triphosphate;
1-alkyl-6-vinyl-pseudo-uridine triphosphate; 1-allylpseudouridine
triphosphate; 1-aminomethyl-pseudo-uridine triphosphate;
1-benzoylpseudouridine triphosphate; 1-benzyloxymethylpseudouridine
triphosphate; 1-benzyl-pseudo-uridine triphosphate;
1-biotinyl-PEG2-pseudouridine triphosphate; 1-bioinylpseudouridine
triphosphate; 1-butyl-pseudo-uridine triphosphate;
1-cyanomethylpseudouridine triphosphate;
1-cyclobutylmethyl-pseudo-uridine triphosphate;
1-cyclobutyl-pseudo-uridine triphosphate;
1-cycloheptylmethyl-pseudo-uridine triphosphate;
1-cycloheptyl-pseudo-uridine triphosphate;
1-cyclohexylmethyl-pseudo-uridine triphosphate;
1-cyclohexyl-pseudo-uridine triphosphate;
1-cyclooctylmethyl-pseudo-uridine triphosphate;
1-cyclooctyl-pseudo-uridine triphosphate;
1-cyclopentylmethyl-pseudo-uridine triphosphate;
1-cyclopentyl-pseudo-uridine triphosphate;
1-cyclopropylmethyl-pseudo-uridine triphosphate;
1-cyclopropyl-pseudo-uridine triphosphate; 1-hexyl-pseudo-uridine
triphosphate; 1-homoallylpseudouridine triphosphate;
1-hydroxymethylpseudouridine triphosphate;
1-iso-propyl-pseudo-uridine triphosphate;
1-me-2-thio-pseudo-uridine triphosphate; 1-me-4-thio-pseudo-uridine
triphosphate; 1-me-alpha-thio-pseudo-uridine triphosphate;
1-methanesulfonylmethylpseudouridine triphosphate;
1-methoxymethylpseudouridine triphosphate;
1-methyl-6-(2,2,2-trifluoroethyl)pseudo-uridine triphosphate;
1-methyl-6-(4-morpholino)-pseudo-uridine triphosphate;
1-methyl-6-(4-thiomorpholino)-pseudo-uridine triphosphate;
1-methyl-6-(substituted phenyl)pseudo-uridine triphosphate;
1-methyl-6-amino-pseudo-uridine triphosphate;
1-methyl-6-azido-pseudo-uridine triphosphate;
1-methyl-6-bromo-pseudo-uridine triphosphate;
1-methyl-6-butyl-pseudo-uridine triphosphate;
1-methyl-6-chloro-pseudo-uridine triphosphate;
1-methyl-6-cyano-pseudo-uridine triphosphate;
1-methyl-6-dimethylamino-pseudo-uridine triphosphate;
1-methyl-6-ethoxy-pseudo-uridine triphosphate;
1-methyl-6-ethylcarboxylate-pseudo-uridine triphosphate;
1-methyl-6-ethyl-pseudo-uridine triphosphate;
1-methyl-6-fluoro-pseudo-uridine triphosphate;
1-methyl-6-formyl-pseudo-uridine triphosphate;
1-methyl-6-hydroxyamino-pseudo-uridine triphosphate;
1-methyl-6-hydroxy-pseudo-uridine triphosphate;
1-methyl-6-iodo-pseudo-uridine triphosphate;
1-methyl-6-iso-propyl-pseudo-uridine triphosphate;
1-methyl-6-methoxy-pseudo-uridine triphosphate;
1-methyl-6-methylamino-pseudo-uridine triphosphate;
1-methyl-6-phenyl-pseudo-uridine triphosphate;
1-methyl-6-propyl-pseudo-uridine triphosphate;
1-methyl-6-tert-butyl-pseudo-uridine triphosphate;
1-methyl-6-trifluoromethoxy-pseudo-uridine triphosphate;
1-methyl-6-trifluoromethyl-pseudo-uridine triphosphate;
1-morpholinomethylpseudouridine triphosphate;
1-pentyl-pseudo-uridine triphosphate; 1-phenyl-pseudo-uridine
triphosphate; 1-pivaloylpseudouridine triphosphate;
1-propargylpseudouridine triphosphate; 1-propyl-pseudo-uridine
triphosphate; 1-propynyl-pseudouridine; 1-p-tolyl-pseudo-uridine
triphosphate; 1-tert-Butyl-pseudo-uridine triphosphate;
1-thiomethoxymethylpseudouridine triphosphate;
1-thiomorpholinomethylpseudouridine triphosphate;
1-trifluoroacetylpseudouridine triphosphate;
1-trifluoromethyl-pseudo-uridine triphosphate; 1-vinylpseudouridine
triphosphate; 2,2'-anhydro-uridine triphosphate;
2'-bromo-deoxyuridine triphosphate; 2'-F-5-methyl-2'-deoxy-uridine
triphosphate; 2'-methoxy-5-methyl-uridine triphosphate;
2'-methoxy-pseudo-uridine triphosphate; 2'-a-ethynyluridine
triphosphate; 2'-a-trifluoromethyluridine triphosphate;
2'-b-ethynyluridine triphosphate; 2'-b-trifluoromethyluridine
triphosphate; 2'-deoxy-2',2'-difluomouridinetriphosphate;
2'-deoxy-2'-a-mercaptouridine triphosphate;
2'-deoxy-2'-a-thiomethoxyuridine triphosphate;
2'-deoxy-2'-b-aminouridine triphosphate; 2'-deoxy-2'-b-azidouridine
triphosphate; 2'-deoxy-2'-b-bromouridine triphosphate;
2'-deoxy-2'-b-chlorouridine triphosphate;
2'-deoxy-2'-b-fluorouridine triphosphate; 2'-deoxy-2'-b-iodouridine
triphosphate; 2'-deoxy-2'-b-mercaptouridine triphosphate;
2'-deoxy-2'-b-thiomethoxyuridine triphosphate;
2-methoxy-4-thio-uridine; 2-methoxyuridine;
2'-O-methyl-5-(1-propynyl)uridine triphosphate;
3-alkyl-pseudo-uridine triphosphate; 4'-azidouridine triphosphate;
4'-carbocyclic uridinetriphosphate; 4'-ethynyluridine triphosphate;
5-(1-propynyl)ara-uridine triphosphate; 5-(2-ruranyl)uridine
triphosphate; 5-cyanouridine triphosphate; 5-dimethylaminouridine
triphosphate; 5'-homo-uridine triphosphate;
5-iodo-2'-fluoro-deoxyuridine triphosphate; 5-phenylethynyluridine
triphosphate; 5-trideuteromethyl-6-deuterouridine triphosphate;
5-trifluoromethyl-uridine triphosphate; 5-vinylarauridine
triphosphate; 6-(2,2,2-trifluoroethyl)-pseudo-uridine triphosphate;
6-(4-morpholino)-pseudo-uridine triphosphate;
6-(4-thiomorpholino)-pseudo-uridine triphosphate;
6-(substituted-phenyl)-pseudo-uridine triphosphate;
6-amino-pseudo-uridine triphosphate; 6-azido-pseudo-uridine
triphosphate; 6-bromo-pseudo-uridine triphosphate;
6-butyl-pseudo-uridine triphosphate; 6-chloro-pseudo-uridine
triphosphate; 6-cyano-pseudo-uridine triphosphate;
6-dimethylamino-pseudo-uridine triphosphate;
6-ethoxy-pseudo-uridine triphosphate;
6-ethylcarboxylate-pseudo-uridine triphosphate;
6-ethyl-pseudo-uridine triphosphate; 6-fluoro-pseudo-uridine
triphosphate; 6-formyl-pseudo-uridine triphosphate;
6-hydroxyamino-pseudo-uridine triphosphate;
6-hydroxy-pseudo-uridine triphosphate; 6-iodo-pseudo-uridine
triphosphate; 6-iso-propyl-pseudo-uridine triphosphate;
6-methoxy-pseudo-uridine triphosphate; 6-methylamino-pseudo-uridine
triphosphate; 6-methyl-pseudo-uridine triphosphate;
6-phenyl-pseudo-uridine triphosphate; 6-propyl-pseudo-uridine
triphosphate; 6-tert-butyl-pseudo-uridine triphosphate;
6-trifluoromethoxy-pseudo-uridine triphosphate;
6-trifluoromethyl-pseudo-uridine triphosphate;
alpha-thio-pseudo-uridine triphosphate; pseudouridine
1-(4-methylbenzenesulfonic acid) triphosphate; pseudouridine
1-(4-methylbenzoic acid) triphosphate; pseudouridine triphosphate
1-[3-(2-ethoxy)]propionic acid; pseudouridine triphosphate
1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy}-ethoxy]propionic acid;
pseudouridine triphosphate
1-[3-{2-(2-[2-{2(2-ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}]propionic
acid; pseudouridine triphosphate
1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionic acid; pseudouridine
triphosphate 1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid;
pseudouridine triphosphate 1-methylphosphonic acid; pseudouridine
triphosphate 1-methylphosphonic acid diethyl ester; pseudo-uridine
triphosphate-N1-3-propionic acid; pseudo-uridine
triphosphate-N1-4-butanoic acid; pseudo-uridine
triphosphate-N1-5-pentanoic acid; pseudo-uridine
triphosphate-N1-6-hexanoic acid; pseudo-uridine
triphosphate-N1-7-heptanoic acid; pseudo-uridine
triphosphate-N1-methyl-p-benzoic acid; pseudo-uridine
triphosphate-N1-p-benzoic acid; wybutosine; hydroxywybutosine;
isowyosine; peroxywybutosine; undermodified hydroxywybutosine;
4-demethylwyosine; 2,6-(diamino)purine;
1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl:
1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;
1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;
1,3,5-(triaza)-2,6-(dioxa)-naphthalene; 2 (amino)purine;
2,4,5-(trimethyl)phenyl; 2' methyl, 2'amino, 2'azido,
2'fluro-cytidine; 2' methyl, 2'amino, 2'azido, 2'fluro-adenine;
2'methyl, 2'amino, 2'azido, 2'fluro-uridine;
2'-amino-2'-deoxyribose; 2-amino-6-chloro-purine; 2-aza-inosinyl;
2'-azido-2'-deoxyribose; 2'fluoro-2'-deoxyribose;
2'-fluoro-modified bases; 2'-O-methyl-ribose;
2-oxo-7-aminopyridopyrimidin-3-yl; 2-oxo-pyridopyrimidine-3-yl;
2-pyridinone; 3 nitropyrrole;
3-(methyl)-7-(propynyl)isocarbostyrilyl;
3-(methyl)isocarbostyrilyl; 4-(fluoro)-6-(methyl)benzimidazole;
4-(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl;
5 nitroindole; 5 substituted pyrimidines;
5-(methyl)isocarbostyrilyl; 5-nitroinidole; 6-(aza)pyrimidine;
6-(azo)thymine; 6-(methyl)-7-(aza)indolyl; 6-chloro-purine;
6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;
7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;
7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;
7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;
7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;
7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;
7-(aza)indolyl;
7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin1-yl;
7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;
7-(guanidiniuinalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;
7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;
7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;
7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;
7-(propynyl)isocarbostyrilyl; 7-(propynyl)isocarbostyrilyl,
7-deaza-inosinyl; 7-substituted
1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-substituted
1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 9-(methyl)-imidizopyridinyl;
aminoindolyl; anthracenyl;
bis-ortho-(aminioalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;
bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;
difluorotolyl; hypoxanthine; imidizopyridinyl; inosinyl;
isocarbostyrilyl; isoguanisine; N2-substituted purines;
N6-methyl-2-amino-purine; N6-substituted purines; N-alkylated
derivative; napthalenyl; nitrobenzimidazolyl; nitroimidazolyl;
nitroindazolyl; nitropyrizolyl; nubularine; 06-substituted purines;
O-alkylated derivative;
ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;
ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; oxoformycin
triphosphate;
para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;
para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; pentacenyl;
phenanthracenyl; phenyl; pyrenyl; pyridopyrimidin-3-yl;
2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-on-3-yl;
pyrrolopyrimidinyl; pyrrolopyrizinyl; stilbenzyl; substituted
1,2,4-triazoles; tetracenyl; tubercidine; xanthine;
xanthosine-5'-triphosphate; 2-thio-zebularine;
5-aza-2-thio-zebularine; 7-deaza-2-amino-purine; pyridin-4-one
ribonucleoside; 2-amino-riboside-triphosphate; formycin A
triphosphate; formycin B triphosphate; pyrrolosine triphosphate;
2'-hydroxyl-ara-adenosine triphosphate; 2'-hydroxyl-ara-cytidine
triphosphate; 2'-hydroxyl-ara-uridine triphosphate;
2'-hydroxyl-ara-guanosine triphosphate;
5-(2-carbomethoxyvinyl)uridine triphosphate; and
N6-(19-amino-pentaoxanonadecyl)adenosine triphosphate.
[0239] In some embodiments, a nucleobase or a modified nucleobase
of the nucleoside comprises a protecting group. Suitable protecting
groups are described above. A skilled artisan will appreciate that
the selection of a protecting group will be dictated by the nature
of the nucleobase or the modified nucleobase. For example, an amine
can be protected by Ac, iBu, Bn, or Bz.
[0240] In another embodiment, Compounds of the Disclosure are any
one or more of the compounds of Table 3.
TABLE-US-00003 TABLE 3 Cpd. No. Structure Name 3-1 ##STR00099##
1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-
5-((((4S,5R)-4,5-dimethyl-2-sulfido-1,3,2- oxathiaphospholan-2-
yl)oxy)methyl)tetrahydrofuran-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione 3-2 ##STR00100##
(2R,3S,5R)-2-((((4S,5R)-4,5-dimethyl-2-
sulfido-1,3,2-oxathiaphospholan-2-
yl)oxy)methyl)-5-(5-methyl-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3- yl benzoate 3-3
##STR00101## (2R,3S,5R)-5-(3-benzoyl-5-methyl-2,4-dioxo-
3,4-dihydropyrimidin-1(2H)-yl)-2-((((4S,5R)-
4,5-dimethyl-2-sulfido-1,3,2- oxathiaphospholan-2-
yl)oxy)methyl)tetrahydrofuran-3-yl benzoate 3-4 ##STR00102##
(4S,5R)-2-(((3aR,4R,6R,6aR)-6-(6-amino-9H-
purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-
d][1,3]dioxol-4-yl)methoxy)-4,5-diphenyl- 1,3,2-oxathiaphospholane
2-sulfide 3-5 ##STR00103## (4S,5R)-2-(((2R,3R,4R,5R)-5-(6-amino-9H-
purin-9-yl)-2-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-
fluorotetrahydrofuran-3-yl)oxy)-4,5-diphenyl-
1,3,2-oxathiaphospholane 2-sulfide 3-6 ##STR00104##
1-((2R,4S,5R)-4-(benzyloxy)-5-((((4S,5R)-4,5-
diphenyl-2-sulfido-1,3,2-oxathiaphospholan-2-
yl)oxy)methyl)tetrahydrofuran-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione 3-7 ##STR00105##
1-((2R,4S,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-
(((4S,5R)-4,5-diphenyl-2-sulfido-1,3,2-
oxathiaphospholan-2-yl)oxy)tetrahydrofuran-2-
yl)-5-methylpyrimidine-2,4(1H,3H)-dione 3-8 ##STR00106##
1-((2R,4S,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-
(((4S,5R)-4,5-diphenyl-2-sulfido-1,3,2-
oxathiaphospholan-2-yl)oxy)tetrahydrofuran-2-
yl)-5-methylpyrimidine-2,4(1H,3H)-dione 3-9 ##STR00107##
1-((2R,4S,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-
(((4S,5R)-4,5-dimethyl-2-sulfido-1,3,2-
oxathiaphospholan-2-yl)oxy)tetrahydrofuran-2-
yl)-5-methylpyrimidine-2,4(1H,3H)-dione 3-10 ##STR00108##
N-(9-((3aR,4R,6R,6aR)-6-((((4S,5R)-4,5-
dimethyl-2-sulfido-1,3,2-oxathiaphospholan-2-
yl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-
d][1,3]dioxol-4-yl)-9H-purin-6-yl)benzamide 3-11 ##STR00109##
N-(9-((3aR,4R,6R,6aR)-2,2-dimethyl-6-
((((5S,7aR)-7a-methyl-5-(prop-1-en-2-yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathia-
phosphol-2-yl)oxy)methyl)tetrahydrofuro[3,4-
d][1,3]dioxol-4-yl)-9H-purin-6-yl)benzamide 3-12 ##STR00110##
N-(9-((3aR,4R,6R,6aR)-2,2-dimethyl-6-
((((3aR,6S)-3a-methyl-6-(prop-1-en-2-yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathia-
phosphol-2-yl)oxy)methyl)tetrahydrofuro[3,4-
d][1,3]dioxol-4-yl)-9H-purin-6-yl)benzamide 3-13 ##STR00111##
N-(9-((3aR,4R,6R,6aR)-2,2-dimethyl-6-
((((3aR,6S)-3a-methyl-6-(prop-1-en-2-yl)-2-
sulfidohexahydrobenzo[d][1,3,2]dithiaphosphol-
2-yl)oxy)methyl)tetrahydrofuro[3,4-
d][1,3]dioxol-4-yl)-9H-purin-6-yl)benzamide 3-14 ##STR00112##
1-((2R,4S,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-
(((5S,7aR)-7a-methyl-5-(prop-1-en-2-yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathia-
phosphol-2-yl)oxy)tetrahydrofuran-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione 3-15 ##STR00113##
1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-
5-((((5S,7aR)-7a-methyl-5-(prop-1-en-2-yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione 3-16 ##STR00114##
1-((2R,4S,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-
(((2S,3aR,5S,7aR)-7a-methyl-5-(prop-1-en-2- yl)-2-sulfidohexahydro-
benzo[d][1,3,2]oxathiaphosphol-2- yl)oxy)tetrahydrofuran-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione 3-17 ##STR00115##
(2S,3aR,5S,7aR)-2-(2-(2-(4-((4-
chlorophenyl)(phenyl)methyl)piperazin-1-
yl)ethoxy)ethoxy)-7a-methyl-5-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-18
##STR00116## 1-((2R,4S,5S)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-4-
(((2S,3aR,5S,7aR)-7a-methyl-5-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)amino)tetrahydrofuran-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione 3-19 ##STR00117##
1-((2R,4S,5R)-5-(((tert- butyldimethylsilyl)oxy)methyl)-4-
(((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)pyrimidine- 2,4(1H,3H)-dione 3-20
##STR00118## 1-((2R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-4-
(((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-5- methylpyrimidine-2,4(1H,3H)-dione
3-21 ##STR00119## 1-((2R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-4-
(((2R,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)pyrimidine- 2,4(1H,3H)-dione 3-22
##STR00120## 1-((2R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-4-
(((2R,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-5- methylpyrimidine-2,4(1H,3H)-dione
3-23 ##STR00121## l-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-
5-((((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en- 2-yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2- yl)pyrimidine-2,4(1H,3H)-dione
3-24 ##STR00122## 1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-
5-((((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en- 2-yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione 3-25 ##STR00123##
1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-
5-((((2R,3aR,6S,7aR)-3a-methyl-6-(prop-1-en- 2-yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2- yl)pyrimidine-2,4(1H,3H)-dione
3-26 ##STR00124## 1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-
5-((((2R,3aR,6S,7aR)-3a-methyl-6-(prop-1-en- 2-yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione 3-27 ##STR00125##
4-amino-1-((2R,4S,5R)-5-(((tert- butyldimethylsilyl)oxy)methyl)-4-
(((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)pyrimidin- 2(1H)-one 3-28
##STR00126## 4-amino-1-((2R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-4-
(((2R,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)pyrimidin- 2(1H)-one 3-29
##STR00127## 4-amino-1-((2R,4S,5R)-4-((tert-
butyldimethylsilyl)oxy)-5-((((2S,3aS,6R,7aS)-
3a-methyl-6-(prop-1-en-2-yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2- yl)pyrimidin-2(1H)-one 3-30
##STR00128## 4-amino-1-((2R,4S,5R)-4-((tert-
butyldimethylsilyl)oxy)-5-((((2R,3aR,6S,7aR)-
3a-methyl-6-(prop-1-en-2-yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2- yl)pyrimidin-2(1H)-one 3-31
##STR00129## (2S,3aS,6R,7aS)-2-(((2R,3S,5R)-5-(6-amino-
9H-purin-9-yl)-2-(((tert-
butyldimethylsilyl)oxy)methyl)tetrahydrofuran-
3-yl)oxy)-3a-methyl-6-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-32
##STR00130## (2S,3aS,6R,7aS)-2-(((2R,3S,5R)-2-(((tert-
butyldimethylsilyl)oxy)methyl)-5-(6-hydroxy-
9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)-3a- methyl-6-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-33
##STR00131## (2R,3aR,6S,7aR)-2-(((2R,3S,5R)-5-(6-amino-
9H-purin-9-yl)-2-(((tert-
butyldimethylsilyl)oxy)methyl)tetrahydrofuran-
3-yl)oxy)-3a-methyl-6-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-34
##STR00132## (2R,3aR,6S,7aR)-2-(((2R,3S,5R)-2-(((tert-
butyldimethylsilyl)oxy)methyl)-5-(6-hydroxy-
9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)-3a- methyl-6-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-35
##STR00133## (2S,3aS,6R,7aS)-2-(((2R,3S,5R)-5-(6-amino-
9H-purin-9-yl)-3-((tert- butyldimethylsilyl)oxy)tetrahydrofuran-2-
yl)methoxy)-3a-methyl-6-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-35
##STR00134## (2S,3aS,6R,7aS)-2-(((2R,3S,5R)-3-((tert-
butyldimethylsilyl)oxy)-5-(6-hydroxy-9H-
purin-9-yl)tetrahydrofuran-2-yl)methoxy)-3a- methyl-6-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-36
##STR00135## (2R,3aR,6S,7aR)-2-(((2R,3S,5R)-5-(6-amino-
9H-purin-9-yl)-3-((tert- butyldimethylsilyl)oxy)tetrahydrofuran-2-
yl)methoxy)-3a-methyl-6-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-37
##STR00136## (2R,3aR,6S,7aR)-2-(((2R,3S,5R)-3-((tert-
butyldimethylsilyl)oxy)-5-(6-hydroxy-9H-
purin-9-yl)tetrahydrofuran-2-yl)methoxy)-3a- methyl-6-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-38
##STR00137## 2-amino-9-((2R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-4-
(((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-1,9-dihydro- 6H-purin-6-one 3-39
##STR00138## 9-((2R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-4-
(((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-2-hydroxy-
1,9-dihydro-6H-purin-6-one 3-40 ##STR00139##
2-amino-9-((2R,4S,5R)-5-(((tert- butyldimethylsilyl)oxy)methyl)-4-
(((2R,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-1,9-dihydro- 6H-purin-6-one 3-41
##STR00140## 9-((2R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-4-
(((2R,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-2-hydroxy-
1,9-dihydro-6H-purin-6-one 3-42 ##STR00141##
2-amino-9-((2R,4S,5R)-4-((tert-
butyldimethylsilyl)oxy)-5-((((2S,3aS,6R,7aS)-
3a-methyl-6-(prop-1-en-2-yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2-yl)-1,9- dihydro-6H-purin-6-one
3-43 ##STR00142## 9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-
5-((((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en- 2-yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2-yl)-2-
hydroxy-1,9-dihydro-6H-purin-6-one 3-44 ##STR00143##
2-amino-9-((2R,4S,5R)-4-((tert-
butyldimethylsilyl)oxy)-5-((((2R,3aR,6S,7aR)-
3a-methyl-6-(prop-1-en-2-yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2-yl)-1,9- dihydro-6H-purin-6-one
3-45 ##STR00144## 9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-
5-((((2R,3aR,6S,7aR)-3a-methyl-6-(prop-1-en- 2-yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2-yl)-2-
hydroxy-1,9-dihydro-6H-purin-6-one 3-46 ##STR00145##
1-((2R,3R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-hydroxy-4-
(((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-5- methylpyrimidine-2,4(1H,3H)-dione
3-47 ##STR00146## 1-((2R,3R,4R,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-fluoro-4-
(((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-5- methylpyrimidine-2,4(1H,3H)-dione
3-48 ##STR00147## 1-((2R,3R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-hydroxy-4-
(((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)pyrimidine- 2,4(1H,3H)-dione 3-49
##STR00148## 1-((2R,3R,4R,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-fluoro-4-
(((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)pyrimidine- 2,4(1H,3H)-dione 3-50
##STR00149## 1-((2R,3R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-hydroxy-4-
(((2R,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-5- methylpyrimidine-2,4(1H,3H)-dione
3-51 ##STR00150## 1-((2R,3R,4R,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-fluoro-4-
(((2R,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-5- methylpyrimidine-2,4(1H,3H)-dione
3-52 ##STR00151## 1-((2R,3R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-hydroxy-4-
(((2R,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)pyrimidine- 2,4(1H,3H)-dione 3-53
##STR00152## 1-((2R,3R,4R,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-fluoro-4-
(((2R,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)pyrimidine- 2,4(1H,3H)-dione 3-54
##STR00153## 1-((2R,3R,4S,5R)-4-((tert-
butyldimethylsilyl)oxy)-3-hydroxy-5-
((((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione 3-55 ##STR00154##
1-((2R,3R,4R,5R)-4-((tert- butyldimethylsilyl)oxy)-3-fluoro-5-
((((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione 3-56 ##STR00155##
1-((2R,3R,4S,5R)-4-((tert- butyldimethylsilyl)oxy)-3-hydroxy-5-
((((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2- yl)pyrimidine-2,4(1H,3H)-dione
3-57 ##STR00156## 1-((2R,3R,4R,5R)-4-((tert-
butyldimethylsilyl)oxy)-3-fluoro-5-
((((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2- yl)pyrimidine-2,4(1H,3H)-dione
3-58 ##STR00157## 1-((2R,3R,4S,5R)-4-((tert-
butyldimethylsilyl)oxy)-3-hydroxy-5-
((((2R,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione 3-59 ##STR00158##
1-((2R,3R,4R,5R)-4-((tert- butyldimethylsilyl)oxy)-3-fluoro-5-
((((2R,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione 3-60 ##STR00159##
1-((2R,3R,4S,5R)-4-((tert- butyldimethylsilyl)oxy)-3-hydroxy-5-
((((2R,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2- yl)pyrimidine-2,4(1H,3H)-dione
3-61 ##STR00160## 1-((2R,3R,4R,5R)-4-((tert-
butyldimethylsilyl)oxy)-3-fluoro-5-
((((2R,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2- yl)pyrimidine-2,4(1H,3H)-dione
3-62 ##STR00161## 4-amino-1-((2R,3R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-hydroxy-4-
(((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)pyrimidin- 2(1H)-one 3-63
##STR00162## 4-amino-1-((2R,3R,4R,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-fluoro-4-
(((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)pyrimidin- 2(1H)-one 3-64
##STR00163## 4-amino-1-((2R,3R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-hydroxy-4-
(((2R,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)pyrimidin- 2(1H)-one 3-65
##STR00164## 4-amino-1-((2R,3R,4R,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-fluoro-4-
(((2R,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)pyrimidin- 2(1H)-one 3-66
##STR00165## 4-amino-1-((2R,3R,4S,5R)-4-((tert-
butyldimethylsilyl)oxy)-3-hydroxy-5-
((((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2- yl)pyrimidin-2(1H)-one 3-67
##STR00166## 4-amino-1-((2R,3R,4R,5R)-4-((tert-
butyldimethylsilyl)oxy)-3-fluoro-5-
((((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2- yl)pyrimidin-2(1H)-one 3-68
##STR00167## 4-amino-1-((2R,3R,4S,5R)-4-((tert-
butyldimethylsilyl)oxy)-3-hydroxy-5-
((((2R,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2- yl)pyrimidin-2(1H)-one 3-69
##STR00168## 4-amino-1-((2R,3R,4R,5R)-4-((tert-
butyldimethylsilyl)oxy)-3-fluoro-5-
((((2R,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2- yl)pyrimidin-2(1H)-one 3-70
##STR00169## (2S,3aS,6R,7aS)-2-(((2R,3S,4R,5R)-5-(6-
amino-9H-purin-9-yl)-2-(((tert- butyldimethylsilyl)oxy)methyl)-4-
hydroxytetrahydrofuran-3-yl)oxy)-3a-methyl-6- (prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-71
##STR00170## (2S,3aS,6R,7aS)-2-(((2R,3R,4R,5R)-5-(6-
amino-9H-purin-9-yl)-2-(((tert- butyldimethylsilyl)oxy)methyl)-4-
hydroxytetrahydrofuran-3-yl)oxy)-3a-methyl-6- (prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-72
##STR00171## (2S,3aS,6R,7aS)-2-(((2R,3S,4R,5R)-2-(((tert-
butyldimethylsilyl)oxy)methyl)-4-hydroxy-5-
(6-hydroxy-9H-purin-9-yl)tetrahydrofuran-3-
yl)oxy)-3a-methyl-6-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-73
##STR00172## (2S,3aS,6R,7aS)-2-(((2R,3R,4R,5R)-2-(((tert-
butyldimethylsilyl)oxy)methyl)-4-fluoro-5-(6-
hydroxy-9H-purin-9-yl)tetrahydrofuran-3-
yl)oxy)-3a-methyl-6-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-74
##STR00173## (2R,3aR,6S,7aR)-2-(((2R,3S,4R,5R)-5-(6-
amino-9H-purin-9-yl)-2-(((tert- butyldimethylsilyl)oxy)methyl)-4-
hydroxytetrahydrofuran-3-yl)oxy)-3a-methyl-6- (prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-75
##STR00174## (2R,3aR,6S,7aR)-2-(((2R,3R,4R,5R)-5-(6-
amino-9H-purin-9-yl)-2-(((tert- butyldimethylsilyl)oxy)methyl)-4-
fluorotetrahydrofuran-3-yl)oxy)-3a-methyl-6- (prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-76
##STR00175## (2R,3aR,6S,7aR)-2-(((2R,3S,4R,5R)-2-(((tert-
butyldimethylsilyl)oxy)methyl)-4-hydroxy-5-
(6-hydroxy-9H-purin-9-yl)tetrahydrofuran-3-
yl)oxy)-3a-methyl-6-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-77
##STR00176## (2R,3aR,6S,7aR)-2-(((2R,3R,4R,5R)-2-(((tert-
butyldimethylsilyl)oxy)methyl)-4-fluoro-5-(6-
hydroxy-9H-purin-9-yl)tetrahydrofuran-3-
yl)oxy)-3a-methyl-6-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-78
##STR00177## (2S,3aS,6R,7aS)-2-(((2R,3S,4R,5R)-5-(6-
amino-9H-purin-9-yl)-3-((tert- butyldimethylsilyl)oxy)-4-
hydroxytetrahydrofuran-2-yl)methoxy)-3a- methyl-6-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-79
##STR00178## (2S,3aS,6R,7aS)-2-(((2R,3R,4R,5R)-5-(6-
amino-9H-purin-9-yl)-3-((tert- butyldimethylsilyl)oxy)-4-
fluorotetrahydrofuran-2-yl)methoxy)-3a- methyl-6-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-78
##STR00179## (2S,3aS,6R,7aS)-2-(((2R,3S,4R,5R)-3-((tert-
butyldimethylsilyl)oxy)-4-hydroxy-5-(6-
hydroxy-9H-purin-9-yl)tetrahydrofuran-2-
yl)methoxy)-3a-methyl-6-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-79
##STR00180## (2S,3aS,6R,7aS)-2-(((2R,3R,4R,5R)-3-((tert-
butyldimethylsilyl)oxy)-4-fluoro-5-(6-hydroxy-
9H-purin-9-yl)tetrahydrofuran-2-yl)methoxy)-
3a-methyl-6-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-80
##STR00181## (2R,3aR,6S,7aR)-2-(((2R,3S,4R,5R)-5-(6-
amino-9H-purin-9-yl)-3-((tert- butyldimethylsilyl)oxy)-4-
hydroxytetrahydrofuran-2-yl)methoxy)-3a- methyl-6-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-81
##STR00182## (2R,3aR,6S,7aR)-2-(((2R,3R,4R,5R)-5-(6-
amino-9H-purin-9-yl)-3-((tert- butyldimethylsilyl)oxy)-4-
fluorotetrahydrofuran-2-yl)methoxy)-3a- methyl-6-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-82
##STR00183## (2R,3aR,6S,7aR)-2-(((2R,3R,4R,5R)-3-((tert-
butyldimethylsilyl)oxy)-4-fluoro-5-(6-hydroxy-
9H-purin-9-yl)tetrahydrofuran-2-yl)methoxy)-
3a-methyl-6-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-83
##STR00184## (2R,3aR,6S,7aR)-2-(((2R,3S,4R,5R)-3-((tert-
butyldimethylsilyl)oxy)-4-hydroxy-5-(6-
hydroxy-9H-purin-9-yl)tetrahydrofuran-2-
yl)methoxy)-3a-methyl-6-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-84
##STR00185## 2-amino-9-((2R,3R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-hydroxy-4-
(((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-1,9-dihydro- 6H-purin-6-one 3-85
##STR00186## 9-((2R,3R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-hydroxy-4-
(((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-2-hydroxy-
1,9-dihydro-6H-purin-6-one 3-86 ##STR00187##
9-((2R,3R,4R,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-fluoro-4-
(((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-2-hydroxy-
1,9-dihydro-6H-purin-6-one 3-87 ##STR00188##
2-amino-9-((2R,3R,4R,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-fluoro-4-
(((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-1,9-dihydro- 6H-purin-6-one 3-88
##STR00189## 2-amino-9-((2R,3R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-hydroxy-4-
(((2R,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-1,9-dihydro- 6H-purin-6-one 3-89
##STR00190## 2-amino-9-((2R,3R,4R,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-fluoro-4-
(((2R,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-1,9-dihydro- 6H-purin-6-one 3-90
##STR00191## 9-((2R,3R,4R,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-fluoro-4-
(((2R,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-2-hydroxy-
1,9-dihydro-6H-purin-6-one 3-91 ##STR00192##
9-((2R,3R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-hydroxy-4-
(((2R,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-2-hydroxy-
1,9-dihydro-6H-purin-6-one 3-92 ##STR00193##
2-amino-9-((2R,3R,4S,5R)-4-((tert-
butyldimethylsilyl)oxy)-3-hydroxy-5-
((((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2-yl)-1,9- dihydro-6H-purin-6-one
3-93 ##STR00194## 2-amino-9-((2R,3R,4R,5R)-4-((tert-
butyldimethylsilyl)oxy)-3-fluoro-5-
((((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2-yl)-1,9- dihydro-6H-purin-6-one
3-94 ##STR00195## 9-((2R,3R,4R,5R)-4-((tert-
butyldimethylsilyl)oxy)-3-fluoro-5-
((((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2-yl)-2-
hydroxy-1,9-dihydro-6H-purin-6-one 3-95 ##STR00196##
9-((2R,3R,4S,5R)-4-((tert- butyldimethylsilyl)oxy)-3-hydroxy-5-
((((2S,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2-yl)-2-
hydroxy-1,9-dihydro-6H-purin-6-one 3-96 ##STR00197##
2-amino-9-((2R,3R,4S,5R)-4-((tert-
butyldimethylsilyl)oxy)-3-hydroxy-5-
((((2R,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2-yl)-1,9- dihydro-6H-purin-6-one
3-97 ##STR00198## 2-amino-9-((2R,3R,4R,5R)-4-((tert-
butyldimethylsilyl)oxy)-3-fluoro-5-
((((2R,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2-yl)-1,9- dihydro-6H-purin-6-one
3-98 ##STR00199## 9-((2R,3R,4R,5R)-4-((tert-
butyldimethylsilyl)oxy)-3-fluoro-5-
((((2R,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2-yl)-2-
hydroxy-1,9-dihydro-6H-purin-6-one 3-99 ##STR00200##
9-((2R,3R,4S,5R)-4-((tert- butyldimethylsilyl)oxy)-3-hydroxy-5-
((((2R,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2-yl)-2-
hydroxy-1,9-dihydro-6H-purin-6-one 3-100 ##STR00201##
O-((2R,3S,5R)-2-(((tert-
butyldimethylsilyl)oxy)methyl)-5-(5-methyl-
2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)tetrahydrofuran-3-yl)
O-hydrogen (S)- methylphosphonothioate 3-101 ##STR00202##
Thymidine-5-(((tert-butyldimethylsilyl)oxy)-3-
[(Rp)-.alpha.-thio]-triphosphate 3-102 ##STR00203##
Thymidine-5-(((tert-butyldimethylsilyl)oxy)-3-
[(Rp)-.alpha.-thio]-[.gamma.-thio]-triphosphate 3-103 ##STR00204##
Thymidine-5-(((tert-butyldimethylsilyl)oxy)-3-
[(Rp)-.alpha.-thio]-[.beta.-thio]-triphosphate 3-104 ##STR00205##
Thymidine-5-(((tert-butyldimethylsilyl)oxy)-3-
[(Rp)-.alpha.-thio]-[.beta.-thio]-[.gamma.-thio]-triphosphate 3-105
##STR00206## O-((2R,3S,5R)-2-(((tert-
butyldimethylsilyl)oxy)methyl)-5-(5-methyl-
2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)tetrahydrofuran-3-yl)
S-dodecyl O-hydrogen (R)-phosphorodithioate 3-106 ##STR00207##
O-((2R,3S,5R)-2-(((tert-
butyldimethylsilyl)oxy)methyl)-5-(5-methyl-
2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)tetrahydrofuran-3-yl)
O-hydrogen (S)- phosphorofluoridothioate 3-107 ##STR00208##
(2S,3aR,6S,7aR)-2-(((2R,3S,5R)-5-(6-amino-
9H-purin-9-yl)-2-(((tert-
butyldiphenylsilyl)oxy)methyl)tetrahydrofuran-
3-yl)oxy)-3a-methyl-6-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-108
##STR00209## (2R,3aS,6R,7aS)-2-(((2R,3S,5R)-5-(6-amino-
9H-purin-9-yl)-2-(((tert-
butyldiphenylsilyl)oxy)methyl)tetrahydrofuran-
3-yl)oxy)-3a-methyl-6-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-109
##STR00210## 4-amino-1-((2R,4S,5R)-5-(((tert-
butyldiphenylsilyl)oxy)methyl)-4-
(((2S,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)pyrimidin- 2(1H)-one 3-110
##STR00211## 4-amino-1-((2R,4S,5R)-5-(((tert-
butyldiphenylsilyl)oxy)methyl)-4-
(((2R,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)pyrimidin- 2(1H)-one 3-111
##STR00212## 1-((2R,4S,5R)-5-(((tert-
butyldiphenylsilyl)oxy)methyl)-4-
(((2S,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-5- methylpyrimidine-2,4(1H,3H)-dione
3-112 ##STR00213## 1-((2R,4S,5R)-5-(((tert-
butyldiphenylsilyl)oxy)methyl)-4-
(((2R,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-5- methylpyrimidine-2,4(1H,3H)-dione
3-113 ##STR00214## (Z)-N'-(9-((2R,4S,5R)-5-(((tert-
butyldiphenylsilyl)oxy)methyl)-4-
(((2S,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-6-oxo-6,9- dihydro-
lH-purin-2-yl)-N,N- dimethylformimidamide 3-114 ##STR00215##
(Z)-N'-(9-((2R,4S,5R)-5-(((tert- butyldiphenylsilyl)oxy)methyl)-4-
(((2R,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-6-oxo-6,9-
dihydro-1H-purin-2-yl)-N,N- dimethylformimidamide 3-115
##STR00216## 1-((2R,4S,5R)-5-((bis(4-
methoxyphenyl)(phenyl)methoxy)methyl)-4-
(((2R,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-5- methylpyrimidine-2,4(1H,3H)-dione
3-116 ##STR00217## (2S,3aR,6S,7aR)-2-(((2R,3R,4R,5R)-5-(6-
amino-9H-purin-9-yl)-2-(((tert- butyldimethylsilyl)oxy)methyl)-4-
fluorotetrahydrofuran-3-yl)oxy)-3a-methyl-6- (prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-117
##STR00218## (2R,3aS,6R,7aS)-2-(((2R,3R,4R,5R)-5-(6-
amino-9H-purin-9-yl)-2-(((tert- butyldimethylsilyl)oxy)methyl)-4-
fluorotetrahydrofuran-3-yl)oxy)-3a-methyl-6- (prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-118
##STR00219## N-(9-((2R,4S,5R)-5-((bis(4-
methoxyphenyl)(phenyl)methoxy)methyl)-4-
(((2R,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-9H-purin-6- yl)benzamide 3-119
##STR00220## N-(1-((2R,4S,5R)-5-((bis(4-
methoxyphenyl)(phenyl)methoxy)methyl)-4-
(((2R,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-2-oxo-1,2-
dihydropyrimidin-4-yl)benzamide 3-120 ##STR00221##
9-((2R,4S,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-
(((2R,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-2-
(isobutylamino)-1,9-dihydro-6H-purin-6-one 3-121 ##STR00222##
(2R,3aS,6R,7aS)-2-(((2R,3S,5R)-5-(6-amino-
9H-purin-9-yl)-2-(((tert-
butyldimethylsilyl)oxy)methyl)tetrahydrofuran-
3-yl)oxy)-3a-methyl-6-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-122
##STR00223## (2R,3aS,6R,7aS)-2-(((2R,3S,5R)-5-(6-amino-
9H-purin-9-yl)-2-((bis(4-methoxy-
phenyl)(phenyl)methoxy)methyl)tetrahydro-
furan-3-yl)oxy)-3a-methyl-6-(prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-123
##STR00224## O-(((2S,3S,5S)-3-azido-5-(5-methyl-2,4-dioxo-
3,4-dihydropyrimidin-1(2H)- yl)tetrahydrofuran-2-yl)methyl) O,S-
dihydrogen phosphorothioate 3-124 ##STR00225##
(5H-dibenzo[b,f]azepine-5- carbonyl)phosphoramidothioic O,O-acid
3-125 ##STR00226## 1-((2R,4S,5S)-4-azido-5-((((2S,3aR,5S,7aR)-
7a-methyl-5-(prop-1-en-2-yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)methyl)tetrahydrofuran-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione 3-126 ##STR00227##
(2S,3aR,5S,7aR)-2-(((2R,3R,4R,5R)-5-(6-
amino-9H-purin-9-yl)-2-((bis(4-
methoxyphenyl)(phenyl)methoxy)methyl)-4-
fluorotetrahydrofuran-3-yl)oxy)-7a-methyl-5- (prop-1-en-2-
yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide 3-127
##STR00228## 1-((2R,4S,5R)-5-((bis(4-
methoxyphenyl)(phenyl)methoxy)methyl)-4-
((4,5-dimethyl-2-oxido-1,3,2-
oxathiaphospholan-2-yl)oxy)tetrahydrofuran-2-
yl)-5-methylpyrimidine-2,4(1H,3H)-dione 3-128 ##STR00229##
N-(9-((2R,4S,5R)-5-((bis(4-
methoxyphenyl)(phenyl)methoxy)methyl)-4-
(((2S,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-9H-purin-6- yl)benzamide 3-129
##STR00230## N-(1-((2R,4S,5R)-5-((bis(4-
methoxyphenyl)(phenyl)methoxy)methyl)-4-
(((2S,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-5-methyl-2-
oxo-1,2-dihydropyrimidin-4-yl)benzamide 3-130 ##STR00231##
N-(9-((2R,4S,5R)-5-((bis(4-
methoxyphenyl)(phenyl)methoxy)methyl)-4-
(((2S,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-6-oxo-6,9-
dihydro-1H-purin-2-yl)isobutyramide 3-131 ##STR00232##
1-((2R,4S,5R)-5-((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-
(((2S,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2- yl)-2-
sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-
2-yl)oxy)tetrahydrofuran-2-yl)-5- methylpyrimidine-2,4(1H,3H)-dione
3-132 ##STR00233## 1-((2R,4S,5R)-5-((bis(4-
methoxyphenyl)(phenyl)methoxy)methyl)-4-
(((2R,4R,6R)-2-oxido-4,6-dipheny1-1,3,2-
oxathiaphosphinan-2-yl)oxy)tetrahydrofuran-2-
yl)-5-methylpyrimidine-2,4(1H,3H)-dione 3-133 ##STR00234##
1-((2R,4S,5R)-5-(((tert-
butyldiphenylsilyl)oxy)methyl)-4-(((2R,4R,6S)-
2-oxido-4,6-dipheny1-1,3,2-oxathiaphosphinan-
2-yl)oxy)tetrahydrofuran-2-yl)-5- methylpyrimidine-2,4(1H,3H)-dione
3-134 ##STR00235## 1-((2R,4S,5R)-5-(((tert-
butyldiphenylsilyl)oxy)methyl)-4-(((2S,4R,6S)-
2-oxido-4,6-diphenyl-1,3,2-oxathiaphosphinan-
2-yl)oxy)tetrahydrofuran-2-yl)-5- methylpyrimidine-2,4(1H,3H)-dione
3-135 ##STR00236## 1-((2R,4S,5R)-5-(((tert-
butyldiphenylsilyl)oxy)methyl)-4-(((4R,6R)-2-
oxido-4,6-diphenyl-1,3,2-oxathiaphosphinan-2-
yl)oxy)tetrahydrofuran-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione
[0241] In one embodiment, Compounds of the Disclosure are compounds
selected from the group consisting of compounds 3-1 to 3-100,
3-105, 3-106, and 3-123 to 3-127. In another embodiment, Compounds
of the Disclosure are compounds selected from the group consisting
of compounds 3-101 to 3-104 and 3-107 to 3-122. In another
embodiment. Compounds of the Disclosure are compounds selected from
the group consisting of compounds 3-128 to 3-135.
[0242] In yet another embodiment, Compounds of the Disclosure are
compounds represented by Formula (IVa):
##STR00237##
[0243] wherein
[0244] R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are hydrogen;
[0245] X.sup.1 and X.sup.2 are independently O, S or NR.sup.c;
[0246] Y is O, S or NR.sup.c;
[0247] R.sup.c is hydrogen or C.sub.1-C.sub.4 alkyl; and
[0248] Nu is a nucleoside.
[0249] In another embodiment, Compounds of the Disclosure are
compounds represented by Formula (IVa) above: wherein R.sup.1,
R.sup.2, R.sup.3, and R.sup.4 are hydrogen; X.sup.1 and X.sup.2 are
S; and Y is S.
[0250] In another embodiment, Compounds of the Disclosure are any
one or more of the compounds of Table 4.
TABLE-US-00004 TABLE 4 4-1 ##STR00238## 1-((2R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-4-((2-sulfido-
1,3,2-dithiaphospholan-2- yl)oxy)tetrahydrofuran-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione 4-2 ##STR00239##
1-((2R,3R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-hydroxy-4-
((2-sulfido-1,3,2-dithiaphospholan-2-
yl)oxy)tetrahydrofuran-2-yl)-5- methylpyrimidine-2,4(1H,3H)-dione
4-3 ##STR00240## 1-((2R,3R,4R,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-fluoro-4-((2-
sulfido-1,3,2-dithiaphospholan-2- yl)oxy)tetrahydrofuran-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione 4-4 ##STR00241##
1-(2R,3R,4R,5R-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-fluoro-4-((2-
sulfido-1,3,2-dithiaphospholan-2-
yl)oxy)tetrahydrofuran-2-yl)pyrimidine- 2,4(1H,3H)-dione 4-5
##STR00242## 1-((2R,3R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-hydroxy-4-
((2-sulfido-1,3,2-dithiaphospholan-2-
yl)oxy)tetrahydrofuran-2-yl)pyrimidine- 2,4(1H,3H)-dione 4-6
##STR00243## 1-((2R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-4-((2-sulfido-
1,3,2-dithiaphospholan-2- yl)oxy)tetrahydrofuran-2-yl)pyrimidine-
2,4(1H,3H)-dione 4-7 ##STR00244##
1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-
5-(((2-sulfido-1,3,2-dithiaphospholan-2-
yl)oxy)methyl)tetrahydrofuran-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione 4-8 ##STR00245##
1-((2R,3R,4S,5R)-4-((tert-
butyldimethylsilyl)oxy)-3-hydroxy-5-(((2-
sulfido-1,3,2-dithiaphospholan-2-
yl)oxy)methyl)tetrahydrofuran-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione 4-9 ##STR00246##
1-((2R,3R,4R,5R)-4-((tert-
butyldimethylsilyl)oxy)-3-fluoro-5-(((2-sulfido-
1,3,2-dithiaphospholan-2- yl)oxy)methyl)tetrahydrofuran-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione 4-10 ##STR00247##
1-((2R,3R,4R,5R)-4-((tert-
butyldimethylsilyl)oxy)-3-fluoro-5-(((2-sulfido-
1,3,2-dithiaphospholan-2-
yl)oxy)methyl)tetrahydrofuran-2-yl)pyrimidine- 2,4(1H,3H)-dione
4-11 ##STR00248## 1-((2R,3R,4S,5R)-4-((tert-
butyldimethylsilyl)oxy)-3-hydroxy-5-(((2-
sulfido-1,3,2-dithiaphospholan-2-
yl)oxy)methyl)tetrahydrofuran-2-yl)pyrimidine- 2,4(1H,3H)-dione
4-12 ##STR00249## 1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-
5-(((2-sulfido-1,3,2-dithiaphospholan-2-
yl)oxy)methyl)tetrahydrofuran-2-yl)pyrimidine- 2,4(1H,3H)-dione
4-13 ##STR00250## 4-amino-1-((2R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-4-((2-sulfido-
1,3,2-dithiaphospholan-2-
yl)oxy)tetrahydrofuran-2-yl)pyrimidin-2(1H)- one 4-14 ##STR00251##
4-amino-1-((2R,3R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-hydroxy-4-
((2-sulfido-1,3,2-dithiaphospholan-2-
yl)oxy)tetrahydrofuran-2-yl)pyrimidin-2(1H)- one 4-15 ##STR00252##
4-amino-1-((2R,3R,4R,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-fluoro-4-((2-
sulfido-1,3,2-dithiaphospholan-2-
yl)oxy)tetrahydrofuran-2-yl)pyrimidin-2(1H)- one 4-16 ##STR00253##
4-amino-1-((2R,4S,5R)-4-((tert-
butyldimethylsilyl)oxy)-5-(((2-sulfido-1,3,2- dithiaphospholan-2-
yl)oxy)methyl)tetrahydrofuran-2-yl)pyrimidin- 2(1H)-one 4-17
##STR00254## 4-amino-1-((2R,3R,4S,5R)-4-((tert-
butyldimethylsilyl)oxy)-3-hydroxy-5-(((2-
sulfido-1,3,2-dithiaphospholan-2-
yl)oxy)methyl)tetrahydrofuran-2-yl)pyrimidin- 2(1H)-one 4-18
##STR00255## 4-amino-1-((2R,3R,4R,5R)-4-((tert-
butyldimethylsilyl)oxy)-3-fluoro-5-(((2-sulfido-
1,3,2-dithiaphospholan-2-
yl)oxy)methyl)tetrahydrofuran-2-yl)pyrimidin- 2(1H)-one 4-19
##STR00256## 2-(((2R,3S,5R)-5-(6-amino-9H-purin-9-yl)-2- (((tert-
butyldimethylsilyl)oxy)methyl)tetrahydrofuran-
3-yl)oxy)-1,3,2-dithiaphospholane 2-sulfide 4-20 ##STR00257##
2-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-
2-(((tert-butyldimethylsilyl)oxy)methyl)-4-
hydroxytetrahydrofuran-3-yl)oxy)-1,3,2- dithiaphospholane 2-sulfide
4-21 ##STR00258## 2-(((2R,3R,4R,5R)-5-(6-amino-9H-purin-9-yl)-
2-(((tert-butyldimethylsilyl)oxy)methyl)-4-
fluorotetrahydrofuran-3-yl)oxy)-1,3,2- dithiaphospholane 2-sulfide
4-22 ##STR00259## 2-(((2R,3R,4R,5R)-2-(((tert-
butyldimethylsilyl)oxy)methyl)-4-fluoro-5-(6-
hydroxy-9H-purin-9-yl)tetrahydrofuran-3-
yl)oxy)-1,3,2-dithiaphospholane 2-sulfide 4-23 ##STR00260##
2-(((2R,3S,4R,5R)-2-(((tert-
butyldimethylsilyl)oxy)methyl)-4-hydroxy-5-
(6-hydroxy-9H-purin-9-yl)tetrahydrofuran-3-
yl)oxy)-1,3,2-dithiaphospholane 2-sulfide 4-24 ##STR00261##
2-(((2R,3S,5R)-2-(((tert-
butyldimethylsilyl)oxy)methyl)-5-(6-hydroxy-
9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)-1,3,2- dithiaphospholane
2-sulfide 4-25 ##STR00262## 2-(((2R,3S,4R,5R)-3-((tert-
butyldimethylsilyl)oxy)-4-hydroxy-5-(6-
hydroxy-9H-purin-9-yl)tetrahydrofuran-2-
yl)methoxy)-1,3,2-dithiaphospholane 2-sulfide 4-26 ##STR00263##
2-(((2R,3R,4R,5R)-3-((tert-
butyldimethylsilyl)oxy)-4-fluoro-5-(6-hydroxy-
9H-purin-9-yl)tetrahydrofuran-2-yl)methoxy)-
1,3,2-dithiaphospholane 2-sulfide 4-27 ##STR00264##
2-(((2R,3S,5R)-3-((tert- butyldimethylsilyl)oxy)-5-(6-hydroxy-9H-
purin-9-yl)tetrahydrofuran-2-yl)methoxy)- 1,3,2-dithiaphospholane
2-sulfide 4-28 ##STR00265##
2-(((2R,3S,5R)-5-(6-amino-9H-purin-9-yl)-3-
((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-
yl)methoxy)-1,3,2-dithiaphospholane 2-sulfide 4-29 ##STR00266##
2-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-
3-((tert-butyldimethylsilyl)oxy)-4-
hydroxytetrahydrofuran-2-yl)methoxy)-1,3,2- dithiaphospholane
2-sulfide 4-30 ##STR00267##
2-(((2R,3R,4R,5R)-5-(6-amino-9H-purin-9-yl)-
3-((tert-butyldimethylsilyl)oxy)-4-
fluorotetrahydrofuran-2-yl)methoxy)-1,3,2- dithiaphospholane
2-sulfide 4-31 ##STR00268## 2-amino-9-((2R,3R,4R,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-fluoro-4-((2-
sulfido-1,3,2-dithiaphospholan-2-
yl)oxy)tetrahydrofuran-2-yl)-1,9-dihydro-6H- purin-6-one 4-32
##STR00269## 2-amino-9-((2R,3R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-hydroxy-4-
((2-sulfido-1,3,2-dithiaphospholan-2-
yl)oxy)tetrahydrofuran-2-yl)-1,9-dihydro-6H- purin-6-one 4-33
##STR00270## 2-amino-9-((2R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-4-((2-sulfido-
1,3,2-dithiaphospholan-2-
yl)oxy)tetrahydrofuran-2-yl)-1,9-dihydro-6H- purin-6-one 4-34
##STR00271## 9-((2R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-4-((2-sulfido-
1,3,2-dithiaphospholan-2-
yl)oxy)tetrahydrofuran-2-yl)-2-hydroxy-1,9- dihydro-6H-purin-6-one
4-35 ##STR00272## 9-((2R,3R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-hydroxy-4-
((2-sulfido-1,3,2-dithiaphospholan-2-
yl)oxy)tetrahydrofuran-2-yl)-2-hydroxy-1,9- dihydro-6H-purin-6-one
4-36 ##STR00273## 9-((2R,3R,4R,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-3-fluoro-4-((2-
sulfido-1,3,2-dithiaphospholan-2-
yl)oxy)tetrahydrofuran-2-yl)-2-hydroxy-1,9- dihydro-6H-purin-6-one
4-37 ##STR00274## 2-amino-9-((2R,3R,4S,5R)-4-((tert-
butyldimethylsilyl)oxy)-3-hydroxy-5-(((2-
sulfido-1,3,2-dithiaphospholan-2-
yl)oxy)methyl)tetrahydrofuran-2-yl)-1,9- dihydro-6H-purin-6-one
4-38 ##STR00275## 2-amino-9-((2R,3R,4R,5R)-4-((tert-
butyldimethylsilyl)oxy)-3-fluoro-5-(((2-sulfido-
1,3,2-dithiaphospholan-2- yl)oxy)methyl)tetrahydrofuran-2-yl)-1,9-
dihydro-6H-purin-6-one 4-39 ##STR00276##
2-amino-9-((2R,4S,5R)-4-((tert-
butyldimethylsilyl)oxy)-5-(((2-sulfido-1,3,2- dithiaphospholan-2-
yl)oxy)methyl)tetrahydrofuran-2-yl)-1,9- dihydro-6H-purin-6-one
4-40 ##STR00277## 9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-
5-(((2-sulfido-1,3,2-dithiaphospholan-2-
yl)oxy)methyl)tetrahydrofuran-2-yl)-2-
hydroxy-1,9-dihydro-6H-purin-6-one 4-41 ##STR00278##
9-((2R,3R,4R,5R)-4-((tert-
butyldimethylsilyl)oxy)-3-fluoro-5-(((2-sulfido-
1,3,2-dithiaphospholan-2- yl)oxy)methyl)tetrahydrofuran-2-yl)-2-
hydroxy-1,9-dihydro-6H-purin-6-one 4-42 ##STR00279##
9-((2R,3R,4S,5R)-4-((tert-
butyldimethylsilyl)oxy)-3-hydroxy-5-(((2-
sulfido-1,3,2-dithiaphospholan-2-
yl)oxy)methyl)tetrahydrofuran-2-yl)-2-
hydroxy-1,9-dihydro-6H-purin-6-one 4-43 ##STR00280##
1-((2R,4S,5R)-5-(((tert-
butyldiphenylsilyl)oxy)methyl)-4-((2-sulfido-
1,3,2-dithiaphospholan-2- yl)oxy)tetrahydrofuran-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione 4-44 ##STR00281##
N-(1-((2R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-4-((2-sulfido-
1,3,2-dithiaphospholan-2- yl)oxy)tetrahydrofuran-2-yl)-2-oxo-1,2-
dihydropyrimidin-4-yl)benzamide 4-45 ##STR00282##
N-(9-((2R,4S,5R)-5-(((tert-
butyldimethylsilyl)oxy)methyl)-4-((2-sulfido-
1,3,2-dithiaphospholan-2- yl)oxy)tetrahydrofuran-2-yl)-6-oxo-6,9-
dihydro-1H-purin-2-yl)isobutyramide 4-46 ##STR00283##
N-(9-((3aR,4R,6R,6aR)-2,2-dimethyl-6-(((2-
sulfido-1,3,2-dithiaphospholan-2- yl)oxy)methyptetrahydrofuro[3,4-
d][1,3]dioxol-4-yl)-9H-purin-6-yl)benzamide
[0251] In one embodiment, Compounds of the Disclosure are compounds
selected from the group consisting of compounds 4-1 to 4-42 and
compound 4-46. In another embodiment, Compounds of the Disclosure
are compounds selected from the group consisting of compounds 4-43
to 4-45.
Definitions
[0252] Unless otherwise stated, the following terms used in this
application, including the specification and claims, have the
definitions given below. It must be noted that, as used in the
specification and the appended claims, the singular forms "a" "an"
and "the" include plural referents unless the context clearly
dictates otherwise. Unless otherwise indicated, conventional
methods of mass spectroscopy, NMR, HPLC, protein chemistry,
biochemistry, recombinant DNA techniques and pharmacology are
employed. In this application, the use of "or" or "and" means
"and/or" unless stated otherwise. Furthermore, use of the term
"including" as well as other forms, such as "include", "includes"
and "included" is not limiting.
[0253] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure is related. For
example, the Concise Dictionary of Biomedicine and Molecular
Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of
Cell and Molecular Biology, 3rd ed., 1999. Academic Press; and the
Oxford Dictionary of Biochemistry And Molecular Biology, Revised,
2000, Oxford University Press, provide one of skill with a general
dictionary of many of the terms used in this disclosure.
[0254] Wherever aspects are described herein with the language
"comprising," otherwise analogous aspects described in terms of
"consisting of" and/or "consisting essentially of" are also
provided.
[0255] Units, prefixes, and symbols are denoted in their Systeme
International de Unites (SI) accepted form. Numeric ranges are
inclusive of the numbers defining the range. Where a range of
values is recited, it is to be understood that each intervening
integer value, and each fraction thereof, between the recited upper
and lower limits of that range is also specifically disclosed,
along with each subrange between such values. The upper and lower
limits of any range can independently be included in or excluded
from the range, and each range where either, neither or both limits
are included is also encompassed within the invention. Where a
value is explicitly recited, it is to be understood that values
which are about the same quantity or amount as the recited value
are also within the scope of the invention. Where a combination is
disclosed, each subcombination of the elements of that combination
is also specifically disclosed and is within the scope of the
invention. Conversely, where different elements or groups of
elements are individually disclosed, combinations thereof are also
disclosed. Where any element of an invention is disclosed as having
a plurality of alternatives, examples of that invention in which
each alternative is excluded singly or in any combination with the
other alternatives are also hereby disclosed; more than one element
of an invention can have such exclusions, and all combinations of
elements having such exclusions are hereby disclosed.
[0256] The present disclosure is intended to include all isotopes
of atoms occurring in the present compounds. Isotopes include those
atoms having the same atomic number but different mass numbers. By
way of general example and without limitation, isotopes of hydrogen
include deuterium and tritium. The isotopes of hydrogen can be
denoted as .sup.1H (hydrogen), .sup.2H (deuterium) and .sup.3H
(tritium). They are also commonly denoted as D for deuterium and T
for tritium. In the application. CD.sub.3 denotes a methyl group
wherein all of the hydrogen atoms are deuterium. Isotopes of carbon
include .sup.13C and .sup.14C. Isotopically-labeled compounds of
the invention can generally be prepared by conventional techniques
known to those skilled in the art or by processes analogous to
those described herein, using an appropriate isotopically-labeled
reagent in place of the non-labeled reagent otherwise employed.
[0257] In the present disclosure, the term "compound" is meant to
include all stereoisomers and isotopes of the structure depicted.
As used herein, the term "stereoisomer" means any geometric isomer
(e.g., cis- and trans-isomer), enantiomer, or diastereomer of a
compound. The present disclosure encompasses any and all
stereoisomers of the compounds described herein, including
stereomerically pure forms (e.g., geometrically pure,
enantiomerically pure, or diastereomerically pure) and enantiomeric
and stereoisomeric mixtures, e.g., racemates. Enantiomeric and
stereomeric mixtures of compounds and means of resolving them into
their component enantiomers or stereoisomers are well-known.
"Isotopes" refers to atoms having the same atomic number but
different mass numbers resulting from a different number of
neutrons in the nuclei. For example, isotopes of hydrogen include
tritium and deuterium. Further, a compound, salt, or complex of the
present disclosure can be prepared in combination with solvent or
water molecules to form solvates and hydrates by routine
methods.
[0258] In the present disclosure, the term "isomer" means any
tautomer, stereoisomer, enantiomer, or diastereomer of any compound
of the invention. It is recognized that the compounds of the
invention can have one or more chiral centers and/or double bonds
and, therefore, exist as stereoisomers, such as double-bond isomers
(i.e., geometric E/Z isomers) or diastereomers (e.g., enantiomers
(i.e., (+) or (-)) or cis/trans isomers). According to the
invention, the chemical structures depicted herein, and therefore
the compounds of the invention, encompass all of the corresponding
stereoisomers, that is, both the stereomerically pure form (e.g.,
geometrically pure, enantiomerically pure, or diastereomerically
pure) and enantiomeric and stereoisomeric mixtures, e.g.,
racemates. Enantiomeric and stereoisomeric mixtures of compounds of
the invention can typically be resolved into their component
enantiomers or stereoisomers by well-known methods, such as
chiral-phase gas chromatography, chiral-phase high performance
liquid chromatography, crystallizing the compound as a chiral salt
complex, or crystallizing the compound in a chiral solvent.
Enantiomers and stereoisomers can also be obtained from
stereomerically or enantiomerically pure intermediates, reagents,
and catalysts by well-known asymmetric synthetic methods.
[0259] In the present disclosure, the term "stereoisomer" refers to
all possible different isomeric as well as conformational forms
that a compound may possess (e.g., a compound of any formula
described herein), in particular all possible stereochemically and
conformationally isomeric forms, all diastereomers, enantiomers
and/or conformers of the basic molecular structure. Some compounds
of the present disclosure may exist in different tautomeric forms,
all of the latter being included within the scope of the present
disclosure.
[0260] In the present disclosure, the term "enantiomer" means each
individual optically active form of a compound of the invention,
having an optical purity or enantiomeric excess (as determined by
methods standard in the art) of at least 80% (i.e., at least 90% of
one enantiomer and at most 10% of the other enantiomer), at least
90%, or at least 98%.
[0261] In the present disclosure, the term "diastereomer," means
stereoisomers that are not mirror images of one another and are
non-superimposable on one another.
[0262] In the present disclosure, the term "nucleic acid"
encompasses poly- or oligo-ribonucleotides (RNA) and poly- or
oligo-deoxyribonucleotides (DNA); RNA or DNA derived from
N-glycosides or C-glycosides of nucleobases and/or modified
nucleobases; nucleic acids derived from sugars and/or modified
sugars; and nucleic acids derived from phosphate bridges and/or
modified phosphorous-atom bridges. The term encompasses nucleic
acids containing any combinations of nucleobases, modified
nucleobases, sugars, modified sugars, phosphate bridges or modified
phosphorous atom bridges. Examples include, and are not limited to,
nucleic acids containing ribose moieties, nucleic acids containing
deoxyribose moieties, nucleic acids containing both ribose and
deoxyribose moieties, nucleic acids containing ribose and modified
ribose moieties. The prefix "poly-" refers to a nucleic acid
containing about 1 to about 10,000 nucleotide monomer units, and
the prefix "oligo-" refers to a nucleic acid containing about 1 to
about 200 nucleotide monomer units. The term "nucleic acid" can
also encompass CDNs.
[0263] In the present disclosure, the terms "nucleobase" and
"nucleosidic base moiety," used interchangeably, refer to the parts
of nucleic acids that are involved in the hydrogen-bonding that
binds one nucleic acid strand to the complementary strand in a
sequence-specific manner. The most common naturally-occurring
nucleobases are adenine (A), guanine (G), uracil (U), cytosine (C),
and thymine (T).
[0264] In the present disclosure, nucleobases can be represented by
abbreviation A, G, U, C, T, Hyp. Abbreviation A refers to adenine;
G refers to guanine; U refers to uracil; C refers to cytosine; T
refers to thymine; Hyp refers to hypoxanthine.
[0265] In the present disclosure, the terms "modified nucleobase"
and "modified nucleosidic base moiety," used interchangeably, refer
to a moiety that can replace a nucleobase. The modified nucleobase
mimics the spatial arrangement, electronic properties, or some
other physicochemical property of the nucleobase and retains the
property of hydrogen-bonding that binds one nucleic acid strand to
another in a sequence-specific manner. A modified nucleobase
generally can pair with naturally occurring bases (e.g., uracil,
thymine, adenine, cytosine, guanine) without substantially
affecting the melting behavior, recognition by intracellular
enzymes or activity of the oligonucleotide duplex. The terms
"modified nucleobase" and "modified nucleosidic base moiety," used
interchangeably, is further intended to include heterocyclic
compounds that can serve as nucleosidic bases, including certain
`universal bases` that are not nucleosidic bases in the most
classical sense but serve as nucleosidic bases. Especially
mentioned as a universal base is 3-nitropyrrole.
[0266] In the present disclosure, the term "nucleoside" refers to a
compound, glycosylamine, wherein a nucleobase (a nitrogenous base,
such as adenine, guanine, thymine, uracil, 5-methyluracil, etc.) or
a modified nucleobase is covalently bound to a five-carbon sugar
(ribose or deoxyribose) or a modified sugar.
[0267] In the present disclosure, the term "sugar" refers to a
monosaccharide in closed and/or open form. Sugars include, but are
not limited to, ribose, deoxyribose, pentofuranose, pentopyranose,
morpholinos, carbocyclic analogs, hexopyranose moieties and
bicyclic sugars such as those found in locked nucleic acids.
Examples of locked nucleic acids include, without limitation, those
disclosed in WO2016/079181. Examples of Compounds of the Disclosure
(IV) having modified sugars include, without limitation, the
following:
##STR00284## ##STR00285## ##STR00286##
[0268] wherein B is the Base.
[0269] In the present disclosure, the term "modified sugar" refers
to a moiety that can replace a sugar. The modified sugar mimics the
spatial arrangement, electronic properties, or some other
physicochemical property of a sugar.
[0270] In the present disclosure, the term "nucleotide" refers to a
moiety wherein a nucleobase or a modified nucleobase is covalently
linked to a sugar or modified sugar, and the sugar or modified
sugar is covalently linked to a phosphate group or a modified
phosphorous-atom moiety, such a thiophosphate group.
[0271] In the present disclosure, the term "peptide" refers to a
chain of amino acid monomers linked by a peptide bond. Generally, a
peptide will have no more than about 50 amino acids. The term
"peptide" encompasses both naturally and non-naturally occurring
amino acids. A peptide can be linear or cyclic.
[0272] In the present disclosure, the term "protein" comprises one
or more polypeptides arranged in a biologically-functional way.
Examples of biologically-functional proteins include, but are not
limited to, enzymes, antibodies, cytokines, hormones,
trans-membrane proteins, etc.
[0273] In the present disclosure, the term "moiety" refers to a
specific segment or functional group of a molecule. Chemical
moieties are often recognized chemical entities embedded in or
appended to a molecule.
[0274] In the present disclosure, the terms "solid-support" or
"resin," used herein interchangeably, refer to any support which
enables synthetic mass production of nucleic acids and/or peptides
and can be reutilized at need. As used herein, the terms refer to a
polymer that is insoluble in the media employed in the reaction
steps performed to synthesize nucleic acids and/or peptides, and is
derivatized to comprise reactive groups.
[0275] In the present disclosure, the term "linking moiety" refers
to any moiety optionally positioned between the terminal nucleoside
and the solid support or between the terminal nucleoside and
another nucleoside, nucleotide, or nucleic acid.
[0276] In the present disclosure, the term "purified," when used in
relation to nucleic acids, refers to one that is separated from at
least one contaminant. As used herein, a "contaminant" is any
substance that makes another unfit, impure or inferior. Thus, a
purified oligonucleotide is present in a form or setting different
from that which existed prior to subjecting it to a purification
method.
[0277] In the present disclosure, the term "alkyl" as used by
itself or as part of another group refers to unsubstituted
straight- or branched-chain aliphatic hydrocarbons containing one
to twelve carbon atoms, i.e., C.sub.1-12 alkyl, or the number of
carbon atoms designated, e.g., a C.sub.1 alkyl such as methyl, a
C.sub.2 alkyl such as ethyl, a C.sub.3 alkyl such as propyl or
isopropyl, and so on. In one embodiment, the alkyl group is a
C.sub.1-10 alkyl. In another embodiment, the alkyl group is a
C.sub.1-6 alkyl. In another embodiment, the alkyl group is a
C.sub.1-4 alkyl. Non-limiting exemplary C.sub.1-10 alkyl groups
include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl,
tert-butyl, iso-butyl, 3-pentyl, hexyl, heptyl, octyl, nonyl, and
decyl. Non-limiting exemplary C.sub.1-6 alkyl groups include
methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl,
iso-butyl, and hexyl. Non-limiting exemplary C.sub.1-4 alkyl groups
include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl,
tert-butyl, and iso-butyl.
[0278] In the present disclosure, the term "optionally substituted
alkyl" is an alkyl group as defined above, that has one or more of
R.sup.a groups.
[0279] In the present disclosure, the term "cycloalkyl" as used by
itself or as part of another group refers to unsubstituted
saturated and partially unsaturated, e.g., containing one or two
double bonds, cyclic aliphatic hydrocarbons containing one to three
rings having from three to twelve carbon atoms, i.e., C.sub.3-12
cycloalkyl, or the number of carbons designated. In one embodiment,
the cycloalkyl group has two rings. In one embodiment, the
cycloalkyl group has one ring. In another embodiment, the
cycloalkyl is saturated. In another embodiment, the cycloalkyl is
unsaturated. In another embodiment, the cycloalkyl group is a
C.sub.3-8 cycloalkyl group. In another embodiment, the cycloalkyl
group is a C.sub.3-7 cycloalkyl group. In another embodiment, the
cycloalkyl group is a C.sub.5-7 cycloalkyl group. In another
embodiment, the cycloalkyl group is a C.sub.3-6 cycloalkyl group.
The term "cycloalkyl" includes groups wherein a ring --CH.sub.2--
is replaced with a --C(.dbd.O)--. Non-limiting exemplary cycloalkyl
groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
cycloheptyl, cyclooctyl, norbornyl, decalin, adamantyl,
cyclohexenyl, cyclopentenyl, cyclohexenyl, and cyclopentanone.
[0280] In the present disclosure, the term "optionally substituted
cycloalkyl" is a cycloalkyl group as defined above, that has one or
more of R.sup.a groups.
[0281] The term optionally substituted cycloalkyl includes
cycloalkyl groups having a fused optionally substituted aryl, e.g.,
phenyl, or fused optionally substituted heteroaryl, e.g., pyridyl.
An optionally substituted cycloalkyl having a fused optionally
substituted aryl or fused optionally substituted heteroaryl group
may be attached to the remainder of the molecule at any available
carbon atom on the cycloalkyl ring. In one embodiment, the
optionally substituted cycloalkyl group is a 5-, 6-, or 7-membered
cycloalkyl group having a fused phenyl group, wherein the the
phenyl optionally substituted with one, two, or three
substituents.
[0282] In the present disclosure, the term "alkenyl" as used by
itself or as part of another group refers to an alkyl containing
one, two or three carbon-to-carbon double bonds. In one embodiment,
the alkenyl group is a C.sub.2-6 alkenyl group. In another
embodiment, the alkenyl group is a C.sub.2-4 alkenyl group.
Non-limiting exemplary alkenyl groups include ethenyl, propenyl,
isopropenyl, butenyl, sec-butenyl, pentenyl, and hexenyl.
[0283] In the present disclosure, the term "optionally substituted
alkenyl" as used herein by itself or as part of another group
refers to an alkenyl that is either unsubstituted or substituted
with one, two or three substituents independently selected from the
group consisting of halo, nitro, cyano, hydroxy, amino, alkylamino,
dialkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy,
aralkyloxy, alkylthio, carboxamido, sulfonamido, alkylcarbonyl,
arylcarbonyl, alkylsulfonyl, arylsulfonyl, carboxy, carboxyalkyl,
alkyl, optionally substituted cycloalkyl, alkenyl, alkynyl,
optionally substituted aryl, heteroaryl, and optionally substituted
heterocyclo.
[0284] In the present disclosure, the term "alkynyl" as used by
itself or as part of another group refers to an alkyl containing
one to three carbon-to-carbon triple bonds. In one embodiment, the
alkynyl has one carbon-to-carbon triple bond. In one embodiment,
the alkynyl group is a C.sub.2-4 alkynyl group. In another
embodiment, the alkynyl group is a C.sub.2-4 alkynyl group.
Non-limiting exemplary alkynyl groups include ethynyl, propynyl,
butynyl, 2-butynyl, pentynyl, and hexynyl groups.
[0285] In the present disclosure, the term "optionally substituted
alkynyl" as used herein by itself or as part of another group
refers to an alkynyl that is either unsubstituted or substituted
with one, two or three substituents independently selected from the
group consisting of halo, nitro, cyano, hydroxy, amino, alkylamino,
dialkylamino, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy,
aralkyloxy, alkylthio, carboxamido, sulfonamido, alkylcarbonyl,
arylcarbonyl, alkylsulfonyl, arylsulfonyl, carboxy, carboxyalkyl,
alkyl, cycloalkyl, alkenyl, alkynyl, optionally substituted aryl,
optionally substituted heteroaryl, heterocyclo, and --Si(R).sub.3,
wherein R is selected from the group consisting of alkyl and
aryl.
[0286] In the present disclosure, the term "aryl" as used by itself
or as part of another group refers to unsubstituted monocyclic or
bicyclic aromatic ring systems having from six to fourteen carbon
atoms, i.e., a C.sub.6-14 aryl. Non-limiting exemplary aryl groups
include phenyl (abbreviated as "Ph"), naphthyl, phenanthryl,
anthracyl, indenyl, azulenyl, biphenyl, biphenylenyl, and fluorenyl
groups. In one embodiment, the aryl group is a phenyl or
naphthyl.
[0287] In the present disclosure, the term "optionally substituted
aryl" as used herein by itself or as part of another group refers
to an aryl that is either unsubstituted or substituted with one to
five substituents independently selected from the group consisting
of halo, nitro, cyano, hydroxy, amino, alkylamino, dialkylamino,
haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy,
alkylthio, carboxamido, sulfonamido, alkylcarbonyl, arylcarbonyl,
alkylsulfonyl, arylsulfonyl, carboxy, carboxyalkyl, optionally
substituted alkyl, optionally substituted cycloalkyl, alkenyl,
alkynyl, optionally substituted aryl, optionally substituted
heteroaryl, optionally substituted heterocyclo, (alkoxy)alkyl,
(amino)alkyl, (carboxamido)alkyl, mercaptoalkyl, and
(heterocyclo)alkyl.
[0288] In one embodiment, the optionally substituted aryl is an
optionally substituted phenyl. In one embodiment, the optionally
substituted phenyl has four substituents. In another embodiment,
the optionally substituted phenyl has three substituents. In
another embodiment, the optionally substituted phenyl has two
substituents. In another embodiment, the optionally substituted
phenyl has one substituent. Non-limiting exemplary substituted aryl
groups include 2-methylphenyl, 2-methoxyphenyl, 2-fluorophenyl,
2-chlorophenyl, 2-bromophenyl, 3-methylphenyl, 3-methoxyphenyl,
3-fluorophenyl, 3-chlorophenyl, 4-methylphenyl, 4-ethylphenyl,
4-methoxyphenyl, 4-fluorophenyl, 4-chlorophenyl,
2,6-di-fluorophenyl, 2,6-di-chlorophenyl, 2-methyl,
3-methoxyphenyl, 2-ethyl, 3-methoxyphenyl, 3,4-di-methoxyphenyl,
3,5-di-fluorophenyl 3,5-di-methylphenyl, 3,5-dimethoxy,
4-methylphenyl, 2-fluoro-3-chlorophenyl, and
3-chloro-4-fluorophenyl. The term optionally substituted aryl
includes phenyl groups having fused optionally substituted
cycloalkyl and fused optionally substituted heterocyclo rings. An
optionally substituted aryl having a fused optionally substituted
cycloalkyl and fused optionally substituted heterocycle is attached
to the remainder of the molecule at any available carbon atom on
the aryl ring. Non-limiting examples include:
##STR00287##
[0289] In the present disclosure, the term "aryloxy" as used by
itself or as part of another group refers to an optionally
substituted aryl attached to a terminal oxygen atom. A non-limiting
exemplary aryloxy group is PhO.sup.-.
[0290] In the present disclosure, the term "heterocycle,"
"heterocyclyl," or "heterocyclic group" is intended to mean a
stable 3-, 4-, 5-, 6-, or 7-membered monocyclic or bicyclic or 7-,
8-, 9-, 10-, 11-, 12-, 13-, or 14-membered polycyclic heterocyclic
ring that is saturated, partially unsaturated, or fully
unsaturated, and that contains carbon atoms and 1, 2, 3 or 4
heteroatoms independently selected from the group consisting of N,
O and S; and including any polycyclic group in which any of the
above-defined heterocyclic rings is fused to a benzene ring. The
nitrogen and sulfur heteroatoms may optionally be oxidized (i.e.,
N.fwdarw.O and S(O).sub.p, wherein p is 0, 1 or 2). The nitrogen
atom may be substituted or unsubstituted (i.e., N or NR wherein R
is H or another substituent, if defined). The heterocyclic ring may
be attached to its pendant group at any heteroatom or carbon atom
that results in a stable structure. The heterocyclic rings
described herein may be substituted on carbon or on a nitrogen atom
if the resulting compound is stable. A nitrogen in the heterocycle
may optionally be quaternized. It is preferred that when the total
number of S and O atoms in the heterocycle exceeds 1, then these
heteroatoms are not adjacent to one another. It is preferred that
the total number of S and O atoms in the heterocycle is not more
than 1. When the term "heterocycle" is used, it is intended to
include heteroaryl.
[0291] Examples of heterocycles include, but are not limited to,
acridinyl, azetidinyl, azocinyl, benzimidazolyl, benzofuranyl,
benzothiofuranyl, benzthiophenyl, benzoxazolyl, benzoxazolinyl,
benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl,
benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl,
carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl,
2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran,
furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl,
1H-indazolyl, imidazolopyridinyl, indolenyl, indolinyl,
indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,
isochromanyl, isoindazolyl, isoindolinyl, isoindolyl,
isoquinolinyl, isothiazolyl, isothiazolopyridinyl, isoxazolyl,
isoxazolopyridinyl, methylenedioxyphenyl, morpholinyl,
naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl,
1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl,
1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxazolopyridinyl,
oxazolidinylperimidinyl, oxindolyl, pyrimidinyl, phenanthridinyl,
phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathiinyl,
phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl,
4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl,
pyrazolidinyl, pyrazolinyl, pyrazolopyridinyl, pyrazolyl,
pyridazinyl, pyridooxazolyl, pyridoimidazolyl, pyridothiazolyl,
pyridinyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2-pyrrolidonyl,
2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl,
quinoxalinyl, quinuclidinyl, tetrazolyl, tetrahydrofuranyl,
tetrahydroisoquinolinyl, tetrahydroquinolinyl,
6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl,
1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl,
thienyl, thiazolopyridinyl, thienothiazolyl, thienooxazolyl,
thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl,
1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, and xanthenyl.
Also included are fused ring and spiro compounds containing, for
example, the above heterocycles.
[0292] As used herein, the term "bicyclic heterocycle" or "bicyclic
heterocyclic group" is intended to mean a stable 9- or 10-membered
heterocyclic ring system which contains two fused rings and
consists of carbon atoms and 1, 2, 3, or 4 heteroatoms
independently selected from the group consisting of N, O and S. Of
the two fused rings, one ring is a 5- or 6-membered monocyclic
aromatic ring comprising a 5-membered heteroaryl ring, a 6-membered
heteroaryl ring or a benzo ring, each fused to a second ring. The
second ring is a 5- or 6-membered monocyclic ring which is
saturated, partially unsaturated, or unsaturated, and comprises a
5-membered heterocycle, a 6-membered heterocycle or a carbocycle
(provided the first ring is not benzo when the second ring is a
carbocycle).
[0293] The bicyclic heterocyclic group may be attached to its
pendant group at any heteroatom or carbon atom which results in a
stable structure. The bicyclic heterocyclic group described herein
may be substituted on carbon or on a nitrogen atom if the resulting
compound is stable. It is preferred that when the total number of S
and O atoms in the heterocycle exceeds 1, then these heteroatoms
are not adjacent to one another. It is preferred that the total
number of S and O atoms in the heterocycle is not more than 1.
[0294] Examples of a bicyclic heterocyclic group are, but not
limited to, quinolinyl, isoquinolinyl, phthalazinyl, quinazolinyl,
indolyl, isoindolyl, indolinyl, 1H-indazolyl, benzimidazolyl,
1,2,3,4-tetrahydroquinolinyl, 1,2,3,4-tetrahydroisoquinolinyl,
5,6,7,8-tetrahydro-quinolinyl, 2,3-dihydro-benzofuranyl, chromanyl,
1,2,3,4-tetrahydro-quinoxalinyl and
1,2,3,4-tetrahydro-quinazolinyl.
[0295] As used herein, the term "aromatic heterocyclic group" or
"heteroaryl" is intended to mean stable monocyclic and polycyclic
aromatic hydrocarbons that include at least one heteroatom ring
member such as sulfur, oxygen, or nitrogen. Heteroaryl groups
include, without limitation, pyridyl, pyrimidinyl, pyrazinyl,
pyridazinyl, triazinyl, furyl, quinolyl, isoquinolyl, thienyl,
imidazolyl, thiazolyl, indolyl, pyrroyl, oxazolyl, benzofuryl,
benzothienyl, benzthiazolyl, isoxazolyl, pyrazolyl, triazolyl,
tetrazolyl, indazolyl, 1,2,4-thiadiazolyl, isothiazolyl, purinyl,
carbazolyl, benzimidazolyl, indolinyl, benzodioxolanyl and
benzodioxane. Heteroaryl groups are substituted or unsubstituted.
The nitrogen atom is substituted or unsubstituted (i.e., N or NR
wherein R is H or another substituent, if defined). The nitrogen
and sulfur heteroatoms may optionally be oxidized (i.e., N.fwdarw.O
and S(O).sub.p, wherein p is 0, 1 or 2).
[0296] Bridged rings are also included in the definition of
heterocycle. A bridged ring occurs when one or more, preferably one
to three, atoms (i.e., C, O, N, or S) link two non-adjacent carbon
or nitrogen atoms. Examples of bridged rings include, but are not
limited to, one carbon atom, two carbon atoms, one nitrogen atom,
two nitrogen atoms, and a carbon-nitrogen group. It is noted that a
bridge always converts a monocyclic ring into a tricyclic ring.
When a ring is bridged, the substituents recited for the ring may
also be present on the bridge.
[0297] The term "heterocyclylalkyl" refers to a heterocyclyl or
substituted heterocyclyl bonded to an alkyl group connected to the
core of the compound.
[0298] In the present disclosure, the term "heteroaryl" or
"heteroaromatic" refers to unsubstituted monocyclic and bicyclic
aromatic ring systems having 5 to 14 ring atoms, i.e., a 5- to
14-membered heteroaryl, wherein at least one carbon atom of one of
the rings is replaced with a heteroatom independently selected from
the group consisting of oxygen, nitrogen and sulfur. In one
embodiment, the heteroaryl contains 1, 2, 3, or 4 heteroatoms
independently selected from the group consisting of oxygen,
nitrogen and sulfur. In one embodiment, the heteroaryl has three
heteroatoms. In another embodiment, the heteroaryl has two
heteroatoms. In another embodiment, the heteroaryl has one
heteroatom. In another embodiment, the heteroaryl is a 5- to
10-membered heteroaryl. In another embodiment, the heteroaryl is a
5- or 6-membered heteroaryl. In another embodiment, the heteroaryl
has 5 ring atoms, e.g., thienyl, a 5-membered heteroaryl having
four carbon atoms and one sulfur atom. In another embodiment, the
heteroaryl has 6 ring atoms, e.g., pyridyl, a 6-membered heteroaryl
having five carbon atoms and one nitrogen atom. Non-limiting
exemplary heteroaryl groups include thienyl, benzo[b]thienyl,
naphtho[2,3-b]thienyl, thianthrenyl, furyl, benzofuryl, pyranyl,
isobenzofuranyl, benzooxazonyl, chromenyl, xanthenyl, 2H-pyrrolyl,
pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl,
pyridazinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl,
isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, cinnolinyl,
quinazolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, O-carbolinyl,
phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl,
phenazinyl, thiazolyl, isothiazolyl, phenothiazolyl, isoxazolyl,
furazanyl, and phenoxazinyl. In one embodiment, the heteroaryl is
thienyl (e.g., thien-2-yl and thien-3-yl), furyl (e.g., 2-furyl and
3-furyl), pyrrolyl (e.g., 1H-pyrrol-2-yl and 1H-pyrrol-3-yl),
imidazolyl (e.g., 2H-imidazol-2-yl and 2H-imidazol-4-yl), pyrazolyl
(e.g., 1H-pyrazol-3-yl, 1H-pyrazol-4-yl, and 1H-pyrazol-5-yl),
pyridyl (e.g., pyridin-2-yl, pyridin-3-yl, and pyridin-4-yl),
pyrimidinyl (e.g., pyrimidin-2-yl, pyrimidin-4-yl, and
pyrimidin-5-yl), thiazolyl (e.g., thiazol-2-yl, thiazol-4-yl, and
thiazol-5-yl), isothiazolyl (e.g., isothiazol-3-yl,
isothiazol-4-yl, and isothiazol-5-yl), oxazolyl (e.g., oxazol-2-yl,
oxazol-4-yl, and oxazol-5-yl), isoxazolyl (e.g., isoxazol-3-yl,
isoxazol-4-yl, and isoxazol-5-yl), or indazolyl (e.g.,
1H-indazol-3-yl). The term "heteroaryl" also includes possible
N-oxides. A non-limiting exemplary N-oxide is pyridyl N-oxide.
[0299] In one embodiment, the heteroaryl is a 5- or 6-membered
heteroaryl. In one embodiment, the heteroaryl is a 5-membered
heteroaryl, i.e., the heteroaryl is a monocyclic aromatic ring
system having 5 ring atoms wherein at least one carbon atom of the
ring is replaced with a heteroatom independently selected from
nitrogen, oxygen, and sulfur. Non-limiting exemplary 5-membered
heteroaryl groups include thienyl, furyl, pyrrolyl, oxazolyl,
pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, and isoxazolyl.
[0300] In another embodiment, the heteroaryl is a 6-membered
heteroaryl, e.g., the heteroaryl is a monocyclic aromatic ring
system having 6 ring atoms wherein at least one carbon atom of the
ring is replaced with a nitrogen atom. Non-limiting exemplary
6-membered heteroaryl groups include pyridyl, pyrazinyl,
pyrimidinyl, and pyridazinyl.
[0301] In the present disclosure, the term "optionally substituted
heteroaryl" as used by itself or as part of another group refers to
a heteroaryl that is either unsubstituted or substituted with one
to four substituents, e.g., one or two substituents, independently
selected from the group consisting of halo, nitro, cyano, hydroxy,
amino, alkylamino, dialkylamino, haloalkyl, hydroxyalkyl, alkoxy,
haloalkoxy, aryloxy, aralkyloxy, alkylthio, carboxamido,
sulfonamido, alkylcarbonyl, arylcarbonyl, alkylsulfonyl,
arylsulfonyl, carboxy, carboxyalkyl, optionally substituted alkyl,
optionally substituted cycloalkyl, alkenyl, alkynyl, aryl,
heteroaryl, heterocyclo, (alkoxy)alkyl, (amino)alkyl,
(carboxamido)alkyl, mercaptoalkyl, and (heterocyclo)alkyl. In one
embodiment, the optionally substituted heteroaryl has one
substituent. Any available carbon or nitrogen atom can be
substituted. Non-limiting exemplary substituted heteroaryl groups
include but are not limited to:
##STR00288##
[0302] The term optionally substituted heteroaryl includes
heteroaryl groups having a fused optionally substituted cycloalkyl
or fused optionally substituted heterocyclo group. An optionally
substituted heteroaryl having a fused optionally substituted
cycloalkyl or fused optionally substituted heterocyclo group may be
attached to the remainder of the molecule at any available carbon
atom on the heteroaryl ring. Non-limiting examples include:
##STR00289##
[0303] In the present disclosure, the term "halogen" is intended to
include fluorine, chlorine, bromine and iodine.
[0304] In the present disclosure, the term "internucleoside
linkage" refers to a naturally-occurring or modified linkage
between two adjacent nucleosides in an oligonucleotide or a CDN.
Naturally occurring RNA and DNA contain phosphorodiester
internucleoside linkages. An example of a modified internucleoside
linkage is a phosphorouthioate linkage.
[0305] In the present disclosure, the term "heterochiral nucleic
acids" refers to nucleic acids comprising internuleoside linkages
containing phosphorous atoms in different stereochemical
configurations. By analogy, the term "homochiral nucleic acids"
refers to nucleic acids comprising internuleoside linkages
containing phosphorous atoms in the same stereochemical
configuration.
[0306] In the present disclosure, the term "leaving group" refers
to a compound with a pKa of less than about 10. In some
embodiments, the pKa of a leaving group is less than 7.1. Examples
of a leaving group include, but are not limited to, DBU,
1,5-Diazabicyclo[4.3.0]non-5-ene (DBN), and imidazole,
N-Hydroxytetrachlorophthalimide (TCNHPI), N-Hydroxyphthalimide
(NHPI), N-Hydroxysuccinimide (OSu), Hydroxybenzotriazole (HOBt),
1-Hydroxy-7-azabenzotriazole (HOAt), Oxyma, and Uroninium salts. In
some embodiment, the leaving group is selected from the group
consisting of:
##STR00290##
[0307] In the present disclosure, the term "protecting group"
refers to a group that protects a functional group, such as
alcohol, amine, carbonyl, carboxylic acid, phosphate, terminal
alkyne, etc., from an unwanted chemical reaction. In some
embodiments, the functional group is a nucleophile. Examples of
alcohol protecting groups include, but are not limited to, acetyl
(Ac), benzoyl (Bz), benzyl (Bn), .beta.-methoxyethoxymethyl ether
(MEM), dimethoxytrityl (DMT), methoxymethyl ether (MOM),
methoxytrityl (MMT), p-methoxybenzyl ether (PMB), trimethylsislyl
(TMS), tert-butyldimethylsilyl (TBS), tert-Butyldiphenylsilyl ether
(TBDPS), tri-iso-propylsilyloxymethyl (TOM), trityl (Triphenyl
methyl, Tr), pivaloyl (Piv), and the like. In one embodiment, the
protecting group is the protecting group is 4,4'-dimethoxytrityl.
Examples of amine protecting groups include, but are not limited
to, carbobenzyloxy (Cbz), isobutyryl (iBu), p-methoxybenzyl
carbonyl (MOZ), tert-butylcarbonyl (Boc), acetyl (Ac), benzoyl
(Bz), benzyl (Bn), p-methoxybenzyl (PMB), p-methoxyphenyl (PMP),
tosyl (Ts), and the like. Examples of carbonyl protecting groups
include, but are not limited to, acetals and ketals, acylals,
dithianes, and the like. Examples of carboxylic acid protecting
groups include, but are not limited to, methyl esters, benzyl
esters, tert-butyl esters, silyl esters, orthoesters, oxazoline,
and the like. Examples of phosphate protecting groups include, but
are not limited to, 2-cyanoethyl, methyl and the like. Examples of
terminal alkyne protecting groups include, but are not limited to,
propargyl and silyl groups. In one embodiment, a protecting group
is used to protect a 5'-hydroxy group of a nucleoside used in the
methods of the present disclosure. In one embodiment, the
protecting group is DMT. In another embodiment, a protecting group
is used to protect a nucleobase of a nucleoside used in the methods
of the present disclosure. In some embodiments, the protecting
group is an amine protecting group. In one embodiment, the
protecting group is Ac. In another embodiment, the protecting group
is Bz. In yet another embodiment, the protecting group is iBu.
Methods of Making Compounds of the Disclosure
[0308] In another aspect, the present disclosure provides methods
of making Compounds of the Disclosure of Formulae (I)-(IIIe). In
some embodiments, a method of the present disclosure comprises
reacting a compound of formula (VII):
##STR00291##
[0309] wherein X and Y are independently O or S, and
[0310] Ph is phenyl, optionally substituted with on one or more
groups chosen from linear or branched C.sub.1-C.sub.6 alkyl, aryl,
heteroaryl, halogen, --CN, or --NO.sub.2,
[0311] with an epoxide or an episulfide.
[0312] In some embodiments, the methods of the present disclosure
comprise reacting a compound of formula (VII) with an epoxide or an
episulfide at a temperature of from about 20.degree. C. to about
50.degree. C. In other embodiments, the temperature is from about
30.degree. C. to about 40.degree. C. In one embodiment, the
temperature is about 35.degree. C. In one embodiment, the
temperature is 35.degree. C.
[0313] In some embodiments, the reaction is conducted for from
about 30 min to about 4 hours. In other embodiments, the reaction
is conducted for from about 1 hour to about 2 hours. In one
embodiment, the reaction is conducted for about 1 hour. In one
embodiment, the reaction is conducted for 1 hour.
[0314] The above reaction conditions are exemplary, and are not
meant to be limiting. A skilled artisan will appreciate that the
reaction conditions, such as reaction time and temperature, the
identity and the amounts of the solvents and base, etc., can be
varied according to the methods known in the art. In some
embodiments, a reaction is conducted in an aprotic solvent at a
temperature of from about 0.degree. C. to about 100.degree. C. for
about 10 min to about 48 hours. In some embodiments, the
temperature is from about 10.degree. C. to about 80.degree. C.,
from about 20.degree. C. to about 60.degree. C., from about
25.degree. C. to about 40.degree. C., from about 25.degree. C. to
about 30.degree. C., from about 30.degree. C. to about 40.degree.
C. In some embodiments, the time of a reaction is from about 30 min
to about 40 hours, from about 30 min to about 20 hours, from about
30 min to about 10 hours, from about 30 min to about 5 hours, from
about 30 min to about 3 hours, from about 1 hour to about 30 hours,
from about 2 hours to about 20 hours, from about 3 hours to about
10 hours, from about 4 hours to about 8 hours, from about 5 hours
to about 6 hours.
[0315] In some embodiments, an epoxide is represented by Formula
(VIII):
##STR00292##
[0316] wherein
[0317] (a) R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are are
independently hydrogen, CD.sub.3 or CF.sub.3, linear or branched
C.sub.1-C.sub.20 alkyl, optionally substituted with one or more,
the same or different, R.sup.a groups; linear or branched
C.sub.2-C.sub.12 alkenyl; optionally substituted with one or more,
the same or different, R.sup.a groups; linear or branched
C.sub.2-C.sub.12 alkynyl, optionally substituted with one or more,
the same or different, R.sup.a groups; aryl, optionally substituted
with one or more, the same or different, R.sup.a groups;
heteroaryl, optionally substituted with one or more, the same or
different, R.sup.a groups; heterocyclyl, optionally substituted
with one or more, the same or different, R.sup.a groups; or
C.sub.3-C.sub.8 cycloalkyl, optionally substituted with one or
more, the same or different, R.sup.a groups;
[0318] or
[0319] (b) any of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 together
with the carbons to which they are attached form C.sub.4-C.sub.8
cycloalkyl, optionally substituted with one or more, the same or
different, R.sup.a groups;
[0320] wherein
[0321] R.sup.a is hydrogen, deuterium, tritium; halogen; linear or
branched C.sub.1-C.sub.6 alkyl; OH, CN, CF.sub.3,
O--C.sub.1-C.sub.6 alkyl, O-aryl, O-heteroaryl, O--C.sub.3-C.sub.8
cycloalkyl, O-heterocyclyl, --NR.sup.bR.sup.b, --COOR.sup.b or
--CONR.sup.bR.sup.b linear or branched C.sub.2-C.sub.6 alkenyl,
optionally substituted with linear or branched C.sub.1-C.sub.6
alkyl; or linear or branched C.sub.2-C.sub.6 alkynyl, optionally
substituted with a linear or branched C.sub.1-C.sub.6 alkyl;
[0322] R.sup.b is independently at each occurrence, the same or
different, hydrogen, linear or branched C.sub.1-C.sub.12 alkyl,
aryl, heteroaryl, heterocyclyl or C.sub.3-C.sub.8 cycloalkyl;
[0323] provided that either the carbon bearing the R.sup.1 and
R.sup.2 groups, the carbon bearing the R.sup.3 and R.sup.4 groups,
or both, is chiral.
[0324] In some embodiments, an epoxide is represented by Formula
(IX):
##STR00293##
[0325] wherein
[0326] R.sup.2 and R.sup.4 are independently hydrogen, CD.sub.3 or
CF.sub.3, linear or branched C.sub.1-C.sub.20 alkyl, optionally
substituted with one or more, the same or different, R.sup.a
groups; linear or branched C.sub.2-C.sub.12 alkenyl; optionally
substituted with one or more, the same or different, R.sup.a
groups; linear or branched C.sub.2-C.sub.12 alkynyl, optionally
substituted with one or more, the same or different, R.sup.a
groups; aryl, optionally substituted with one or more, the same or
different, R.sup.a groups; heteroaryl, optionally substituted with
one or more, the same or different, R.sup.a groups; heterocyclyl,
optionally substituted with one or more, the same or different,
R.sup.a groups; or C.sub.3-C.sub.8 cycloalkyl, optionally
substituted with one or more, the same or different, R.sup.a
groups;
[0327] wherein
[0328] R.sup.a is hydrogen, deuterium, tritium; halogen; linear or
branched C.sub.1-C.sub.6 alkyl; OH, CN, CF.sub.3,
O--C.sub.1-C.sub.6 alkyl, O-aryl, O-heteroaryl, O--C.sub.3-C.sub.8
cycloalkyl, O-heterocyclyl, --NR.sup.bR.sup.b, --COOR.sup.b or
--CONR.sup.bR.sup.b linear or branched C.sub.2-C.sub.6 alkenyl,
optionally substituted with linear or branched C.sub.1-C.sub.6
alkyl; or linear or branched C.sub.2-C.sub.6 alkynyl, optionally
substituted with a linear or branched C.sub.1-C.sub.6 alkyl;
[0329] R.sup.b is independently at each occurrence, the same or
different, hydrogen, linear or branched C.sub.1-C.sub.12 alkyl,
aryl, heteroaryl, heterocyclyl or C.sub.3-C.sub.8 cycloalkyl.
[0330] Exemplary epoxides include, but are not limited to, the
following epoxides:
##STR00294## ##STR00295## ##STR00296## ##STR00297## ##STR00298##
##STR00299## ##STR00300## ##STR00301## ##STR00302##
##STR00303##
[0331] Exemplary episulfides include, but are not limited to,
thiirane.
[0332] Exemplary compounds represented by Formula (VII) include,
but are not limited to, the following:
##STR00304## ##STR00305## ##STR00306## ##STR00307##
[0333] A skilled artisan would appreciate that the compounds
represented by formula (VII) can be made by methods known in the
art. For example, the compound can be made by the following
reaction:
##STR00308##
[0334] wherein LG is as defined above. In some embodiment, the
reaction can be conducted at a temperature of from about 20.degree.
C. to about 50.degree. C. A skilled artisan will appreciate that
the reaction conditions, such as reaction time and temperature, the
identity and the amounts of the solvents, etc., can be varied
according to the methods known in the art.
[0335] In other embodiments, Compounds of the Disclosure of
Formulae (I)-(IIIe) can be made by a process comprising reacting a
compound of Formula (VII) with a compound of formula (IXa):
##STR00309##
[0336] wherein
[0337] (a) R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6
are independently hydrogen, deuterium, tritium; CD.sub.3 or
CF.sub.3, linear or branched C.sub.1-C.sub.20 alkyl, optionally
substituted with one or more, the same or different, R.sup.a
groups; linear or branched C.sub.2-C.sub.6 alkenyl, optionally
substituted with one or more, the same or different, R.sup.a
groups; linear or branched C.sub.2-C.sub.6 alkynyl, optionally
substituted with one or more, the same or different, R.sup.a
groups; halogen, --CN, --NO.sub.2; aryl, optionally substituted
with one or more, the same or different, R.sup.a groups;
heteroaryl, optionally substituted with one or more, the same or
different, R.sup.a groups; or C.sub.3-C.sub.8 cycloalkyl,
optionally substituted with one or more, the same or different,
R.sup.a groups;
[0338] or
[0339] (b) any of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and
R.sup.6 together with the carbons to which they are attached form
C.sub.4-C.sub.8 cycloalkyl, optionally substituted with one or
more, the same or different, R.sup.a groups; wherein
[0340] R.sup.a is hydrogen, deuterium, tritium; halogen; linear or
branched C.sub.1-C.sub.6 alkyl; OH, CN, CF.sub.3,
O--C.sub.1-C.sub.6 alkyl, O-aryl, O-heteroaryl, O--C.sub.3-C.sub.8
cycloalkyl, O-heterocyclyl, --NR.sup.bR.sup.b, --COOR.sup.b or
--CONR.sup.bR.sup.b linear or branched C.sub.2-C.sub.6 alkenyl,
optionally substituted with linear or branched C.sub.1-C.sub.6
alkyl; or linear or branched C.sub.2-C.sub.6 alkynyl, optionally
substituted with a linear or branched C.sub.1-C.sub.6 alkyl;
[0341] R.sup.b is independently at each occurrence, the same or
different, hydrogen, linear or branched C.sub.1-C.sub.12 alkyl,
aryl, heteroaryl, heterocyclyl or C.sub.3-C.sub.8 cycloalkyl;
[0342] provided that either the carbon bearing the R.sup.1 or
R.sup.2 groups, the carbon bearing the R.sup.3 or R.sup.4 groups,
or the carbon bearing the R.sup.4 or R.sup.5 groups or all of the
above, is chiral.
[0343] In other embodiments, Compounds of the Disclosure of
Formulae (I)-(IIIe) can be made by a process comprising reacting a
compound of Formula (VII) with a compound of formula (IXb):
##STR00310##
[0344] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and
R.sup.6 are as defined above; and Z is a halogen.
[0345] In another embodiment, Compounds of the Disclosure of
Formulae (I)-(IIIc) can be made by reacting mercaptoethanol or
mercaptopropanol and PCl.sub.3. The mercaptoethanol or
mercaptopropanol can be represented by the following formulae:
##STR00311##
[0346] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, LG are as defined above.
[0347] In one embodiment, Compounds of the Disclosure of Formulae
(I)-(IIIe) can be made according to the following exemplary
reaction scheme:
##STR00312##
[0348] wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 and LG are as
defined above. Additional reagents that can affect the reaction
between a mercaptoethanol and PCl.sub.3 include, but are not
limited to, potassium peroxymonosulfate (Oxone,) magnesium
monoperoxyphthalate (MMPP) and trichloroacetaldehyde.
[0349] In one embodiment, Compounds of the Disclosure of Formulae
(I)-(IIIe) can be made according to the following exemplary
reaction scheme:
##STR00313##
[0350] wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 and LG are as
defined above.
[0351] In some embodiments, the reactions using a mercaptoethanol
are conducted in an aprotic solvent at a temperature of from about
0.degree. C. to about 100.degree. C. for about 10 min to about 48
hours. In some embodiments, the temperature is from about
10.degree. C. to about 80.degree. C., from about 20.degree. C. to
about 60.degree. C., from about 25.degree. C. to about 40.degree.
C., from about 25.degree. C. to about 30.degree. C., from about
30.degree. C. to about 40.degree. C. In some embodiments, the time
of a reaction is from about 30 min to about 40 hours, from about 30
min to about 20 hours, from about 30 min to about 10 hours, from
about 30 min to about 5 hours, from about 30 min to about 3 hours,
from about 1 hour to about 30 hours, from about 2 hours to about 20
hours, from about 3 hours to about 10 hours, from about 4 hours to
about 8 hours, from about 5 hours to about 6 hours.
[0352] In one embodiment, Compounds of the Disclosure of Formulae
(I)-(IIIe) can be made according to the following exemplary
reaction scheme:
##STR00314##
[0353] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and
R.sup.6 are as defined above. In one embodiment, X is a leaving
group (LG) as defined above. In another embodiment, X is --OH,
--Cl, or 4-nitrophenoxy. A base can be varied based on the
conditioin. In some embodiments, the base is chosen from
triethylamine, DBU, DIPEA, pyridine, 2,6-Lut., or imidazole. In
another embodiment, the base is triethylamine.
[0354] The above reaction conditions are exemplary, and are not
meant to be limiting. A skilled artisan will appreciate that the
reaction conditions, such as reaction time and temperature, the
identity and the amounts of the solvents, etc., can be varied
according to the methods known in the art. A skilled artisan will
also be familiar with other solvents (e.g., toluene, p-xylene,
n-hexane, etc.) and sulfurization agents (e.g., the Beaucage
Reagent, K.sub.2S.sub.5O.sub.6, etc.) can be used in the above
reaction.
[0355] Examples of suitable mercaptoethanols include, but are not
limited to, the following:
##STR00315##
[0356] In yet another embodiment, Compounds of the Disclosure can
be made according to the following reaction scheme:
##STR00316##
[0357] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, and LG are as
defined above.
[0358] In some embodiments, a reaction is conducted in an aprotic
solvent at a temperature of from about 0.degree. C. to about
100.degree. C. for about 10 min to about 48 hours. In some
embodiments, the temperature is from about 10.degree. C. to about
80.degree. C., from about 20.degree. C. to about 60.degree. C.,
from about 25.degree. C. to about 40.degree. C., from about
25.degree. C. to about 30.degree. C., from about 30.degree. C. to
about 40.degree. C. In some embodiments, the time of a reaction is
from about 30 min to about 40 hours, from about 30 min to about 20
hours, from about 30 min to about 10 hours, from about 30 min to
about 5 hours, from about 30 min to about 3 hours, from about 1
hour to about 30 hours, from about 2 hours to about 20 hours, from
about 3 hours to about 10 hours, from about 4 hours to about 8
hours, from about 5 hours to about 6 hours.
[0359] The above reaction conditions are exemplary, and are not
meant to be limiting. A skilled artisan will appreciate that the
reaction conditions, such as reaction time and temperature, the
identity and the amounts of the solvents, etc., can be varied
according to the methods known in the art.
[0360] In another aspect, the present disclosure provides methods
for making stereo-defined organosphosphorous (V) compounds. The
stereo-defined organosphosphorous (V) compounds can be made by
coupling an oxathiaphospholane sulfide compound that is designed to
undergo cleavage to a nucleophile, followed by a reaction with
another nucleophile, thereby making a stereo-defined
organophoshorous (V) compound. Examples of suitable nucleophiles
include, but are not limited to, water, hydroxide anions, alcohols,
alkoxide anions, carboxylate anions, thiols, thiolate anions,
anions of a thiocarboxylic acid, amines, amides, etc. The methods
of the present disclosure are not limited to a particular
stereo-defined organophoshorous (V) compound, but can be used to
prepare oligonucleotides; CDNs; conjugates comprising
peptides/proteins and nucleic acids; peptide/protein-drug
conjugates, etc. The nature and the origin of nucleophiles are also
not limited to a particular molecule, but nucleophiles can be
supplied by, for example, nucleic acids (e.g., 3'-hydroxy and
5'-hydroxy), peptides and proteins (e.g., --NH.sub.2, --OH, --SH,
--C(O)NH.sub.2, --C(O)OH, etc.), or small molecules (--NH.sub.2,
--OH, --SH, --C(O)NH.sub.2, --C(O)OH, etc.).
[0361] In some embodiments, Compounds of the Present Disclosure
represented by any one of Formulae (I)-(IIIe) are used to prepare
compounds represented by any one of Formulae (IV)-(VIe), above. In
these embodiments, a method comprises reacting a compound
represented by any one of Formulae (I)-(IIIe), above, with a
nucleoside. In some embodiments, the reaction is conducted in the
presence of a base. In some embodiments, the base is selected from
the group consisting of DBU, BTMG, TMG, LiHMDS, LiOtBu, KHMDS.
KOtBu, NaHMDS, NaOtBu, DABCO, NMI, DIPEA, Pyr, 2,6-Lut, and
imidazole.
[0362] In some embodiments, the reaction is conducted at a
temperature of from about -78.degree. C. to about 30.degree. C. In
other embodiments, the temperature is from about -50.degree. C. to
about 30.degree. C. In other embodiments, the temperature is from
about -30.degree. C. to about 30.degree. C. In other embodiments,
the temperature is from about -10.degree. C. to about 30.degree. C.
In other embodiments, the temperature is from about 0.degree. C. to
about 30.degree. C. In other embodiments, the temperature is from
about 10.degree. C. to about 30.degree. C. In other embodiments,
the temperature is from about 15.degree. C. to about 30.degree. C.
In other embodiments, the temperature is from about 20.degree. C.
to about 30.degree. C. In other embodiments, the temperature is
from about 25.degree. C. to about 30.degree. C. In one embodiment,
the temperature is from about 20.degree. C. In one embodiment, the
temperature is from about 25.degree. C.
[0363] In some embodiments, the reaction is conducted for from
about 10 min to about 12 hours. In other embodiments, the reaction
is conducted for from about 1 hour to about 8 hours. In other
embodiments, the reaction is conducted for from about 1 hour to
about 6 hours. In other embodiments, the reaction is conducted for
from about 1 hour to about 4 hours. In other embodiments, the
reaction is conducted for from about 1 hour to about 2 hours. In
one embodiment, the reaction is conducted for about 1 hour. In one
embodiment, the reaction is conducted for 1 hour.
[0364] As described above the nucleoside may comprise a naturally
occurring or a modified base. It may also comprise a naturally
occurring or a modified sugar, as described above. In some
embodiments, both the sugar and the base are naturally occurring.
In other embodiments, both the sugar and the base are modified. In
yet other embodiments only one (a sugar or a base) are modified. In
one embodiment, the nucleoside is a ribonucleoside. In another
embodiment, the nucleoside is deoxyribonucleoside.
[0365] In one embodiment, the nucleoside comprises a protecting
group at the 5'-end. Suitable protecting groups are described
above. In one embodiment, the protecting group is DMTr. In one
embodiment, the nucleoside comprises a protecting group at the
3'-end. Suitable protecting groups are described above. In one
embodiment, the protecting group is TBS. In another embodiment,
when the nucleoside is a deoxyribonucleoside, it can comprise a
protecting group at the 2'-end. Suitable protecting groups are
described above. In one embodiment, the protecting group is TOM. In
another embodiment, the protecting group is TBDPS.
[0366] In another embodiment, a nucleobase or a modified nucleobase
of the nucleoside comprises a protecting group. Suitable protecting
groups are described above. A skilled artisan will appreciate that
the selection of a protecting group will be dictated by the nature
of the nucleobase or the modified nucleobase. For example, an amine
can be protected by Ac, iBu, or Bz.
[0367] In some embodiments, the protecting group(s) are removed
from the final product. A skilled artisan will be familiar with
methods for removing protecting groups. For example, the DMTr
protecting group can be removed by a weak acid, such as
trichloroacetic acid (TCA, pKa 0.8) or dichloroacetic acid (DCA,
pKa 1.5), in dichloromethane. The TBS, TOM, and TBDPS protecting
groups can be removed by an acid or fluoride ion, such as NaF, TBAF
(tetra-n-butylammonium fluoride, HF.Py, or HF.NEt.sub.3. The Ac,
iBu, and Bz protecting groups can be removed by a base, such as
aqueous or gaseous ammonia or methylamine.
[0368] The above reaction conditions and reagents are exemplary,
and are not meant to be limiting. A skilled artisan will appreciate
that the reaction conditions, such as reaction time and
temperature, the identity and the amounts of the solvents and base,
etc., can be varied according to the methods known in the art.
Methods of Making Oligonucleotides and CDNs Comprising
Stereo-Defined Thio-Phosphate Based Internucleoside Linkages
[0369] In another aspect, the present disclosure provides a method
for making oligonucleotides and CDNs comprising stereo-defined,
enantioenriched thiophosphate-based internucleoside linkages. In
some embodiments, the method comprises (a) reacting a compound of
any of the Formulae (I)-(IIIe) with a nucleoside, to make a
compound of any of the Formulae (IV)-(VIe), as described above, and
(b) reacting the compound formed in step (a) with another
nucleoside, thereby forming an internucleoside linkage.
[0370] In some embodiments, the internucleoside linkage formed by
the method above is stereo-defined. In some embodiment, the
internucleoside linkage formed by the method above is achiral.
[0371] In some embodiments, both reactions are conducted in the
presence of a base. Suitable bases are described above. Reaction
conditions for such reaction are also described above. A skilled
artisan will easily be able to vary the reaction conditions, such
as time, temperature, amounts of the reagents, etc., to achieve
desirable yields.
[0372] In some embodiments, nucleosides can comprise
naturally-occurring or modified nucleobases and sugars. Suitable
naturally-occurring and modified nucleobases and sugars are
described above. In some embodiments, the nucleosides can bear
protecting groups. Suitable sugar and nucleobase protecting groups
are described above. In one embodiment, the nucleosides in the two
reactions are the same. In another embodiment, the nucleosides are
different. In some embodiments, protecting groups can be removed by
the methods described above.
[0373] In some embodiments, the linkage is a phosphorothioate
linkage.
[0374] The method of making stereo-defined thio-phosphate
internucleoside linkages is uniquely applicable for making
stereo-defined CDNs. In this application, the method further
comprises (c) adding a compound of any of the Formulae (I)-(IIIe),
thereby forming a CDN. Exemplary, but not limiting, embodiments of
the method are illustrated below:
##STR00317##
[0375] In some embodiments, the methods of making a CDN comprise a
purification step. Purification of the CDN can be performed by
methods known in the arts such as, but not limited to, HPLC based
purification methods such as, but not limited to, strong anion
exchange HPLC, weak anion exchange HPLC, reverse phase HPLC
(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC).
[0376] The present disclosure also provides a method for preparing
a homochiral thio-phosphate-based oligonucleotide and salts
thereof. In some embodiments, the method comprises:
[0377] a) reacting a compound of any of the Formulae (I)-(IIIe)
with a nucleoside;
[0378] b) reacting the compound formed in step (a) with another
nucleoside, thereby forming an internucleoside linkage (e.g.,
coupling);
[0379] c) adding a compound of any of the Formulae (I)-(IIIe);
[0380] d) adding another nucleoside, thereby coupling said
nucleoside to the growing oligonucleotide; and
[0381] e) repeating steps (c) and (d) until the oligonucleotide
comprises a desired number of nucleotides.
[0382] In some embodiments, both reactions are conducted in the
presence of a base. Suitable bases are described above. Reaction
conditions for such reaction are also described above. A skilled
artisan will easily be able to vary the reaction conditions, such
as time, temperature, amounts of the reagents, etc., to achieve
desirable yields.
[0383] In some embodiments, nucleosides can comprise
naturally-occurring or modified nucleobases and sugars. Suitable
naturally-occurring and modified nucleobases and sugars are
described above. Suitable sugar and nucleobase protecting groups
are described above. It will be within a purview of a skilled
artisan to select appropriate nucleosides, based on the eventual
application of the oligonucleotides.
[0384] In some embodiments, the nucleosides can bear a protecting
group. In some embodiments, the protecting group can be removed by
the methods described above. In one embodiment, the 5'-hydroxy
protecting group is removed after each coupling step. In one
embodiment, the 2'-hydroxy protecting group is not removed until
the oligonucleotide synthesis is complete. In one embodiment, the
nucleobase protecting groups are not removed until the
oligonucleotide synthesis is completed.
[0385] In one embodiment, the oligonucleotide is a homochiral
phosphorothioate-based oligonucleotide.
[0386] Examples of the salts of the oligonucleotide include, but
are not limited to, ammonium salts, such as salts of a tertiary
alkylamine compound (e.g., triethylamine salts), metal salts, such
as sodium salts, potassium salts, and magnesium salts, etc. The
oligonucleotide or a salt thereof may be in the form of a hydrate
or a solvate.
[0387] In certain embodiments, the oligonucleotide can be
synthesized using iterative oligonucleotide synthesis in a
solution.
[0388] In certain embodiments, the oligonucleotide can be
synthesized using iterative solid-phase nucleic acid synthetic
regimes. Typically, the first step in such a process is attachment
of the first nucleoside containing a protected 5'-hydroxyl to a
solid support (also referred to herein as "resin"), usually through
a linker, using standard methods and procedures known in the art.
See, e.g., Oligonucleotides and Analogues: A Practical Approach,
Ekstein, F. Ed., IRL Press, N.Y., 1991, hereby incorporated by
reference in its entirety. The support-bound nucleoside is then
treated to remove the 5'-protecting group, using the methods
described above. The solid support bound nucleoside is then reacted
with a second nucleoside in the presence of a compound according to
any of the Formulae (I)-(IIIe) thereby forming an internuleoside
linkage.
[0389] In some embodiments, the oligonucleotide synthesis is
performed on an automated synthesizer utilizing a solid support.
Solid supports are substrates which are capable of serving as the
solid phase in solid phase synthetic methodologies, such as those
described in U.S. Pat. Nos. 4,415,732; 4,458,066; 4,500,707;
4,668,777; 4,973,679; 5,132,418; 4,725,677 and Re. 34,069. Linkers
are known in the art as short molecules which serve to connect a
solid support to functional groups, e.g., hydroxyl groups, of
initial synthon molecules in solid phase synthetic techniques.
Suitable linkers are disclosed in, for example. Oligonucleotides
and Analogues: A Practical Approach, Ecstein, F., Ed., IRL Press,
N.Y., 1991, Chapter 1, pages 1-23.
[0390] Suitable solid support also includes those generally known
in the art to be suitable for use in solid-phase methodologies
including, for example, controlled pore glass (CPG),
oxalyl-controlled pore glass (see, e.g., Alul et al., Nucleic Acids
Research 1991, 19, 1527, hereby incorporated by reference in its
entirety), TentaGel Support; an aminopolyethyleneglycol derivatized
support (see, e.g., Wright et al., Tetrahedron Letters 1993, 34,
3373, hereby incorporated by reference in its entirety) and Poros;
a copolymer of polystyrene/divinylbenzene.
[0391] In some embodiments, the oligonucleotide can be made
manually. In other embodiments, the oligonucleotides can be made on
an automated synthesizer. In one embodiment, the synthesizer is an
automated solid-phase peptide synthesizer. In one embodiment, the
synthesizer is an automated solid-phase oligonucleotide
synthesizer.
[0392] The extension of the oligonucleotide can be performed in the
3' to 5' direction. In one embodiment, the oligonucleotide is
synthesized from the free hydroxyl at the 5'-end in repetitive
cycles of chemical reactions. Alternatively, the extension of the
oligonucleotide can be performed in the 5' to 3' direction. In an
embodiment, the oligonucleotide is synthesized from the free
hydroxyl at the 3'-end in repetitive cycles of chemical
reactions.
[0393] In some embodiments, the method of making an oligonucleotide
comprises a purification step. Purification can be performed by
methods known in the arts such as, but not limited to, AGENCOURT@
beads (Beckman Coulter Genomics, Danvers, Mass.), poly-T beads,
LNA.TM. oligo-T capture probes (EXIQON.RTM. Inc., Vedbaek, Denmark)
or HPLC based purification methods such as, but not limited to,
strong anion exchange HPLC, weak anion exchange HPLC, reverse phase
HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC).
[0394] In some embodiments, a method of synthesizing nucleic acids
is carried out according to the scheme in FIG. 2.
[0395] In some embodiments, the completed oligonucleotide is
cleaved from the solid support. The cleavage step can precede or
follow deprotection of protected functional groups. In certain
embodiments, the oligonucleotide is left attached on the solid
support for purification purposes and then cleaved from the solid
support following purification.
[0396] In some embodiments, a non-hydrolyzable cap structure can be
added to an oligonucleotide. Because cap structure hydrolysis
requires cleavage of 5'-ppp-5' phosphorodiester linkages, modified
nucleotides can be used during the capping reaction. For example, a
Vaccinia Capping Enzyme from New England Biolabs (Ipswich, Mass.)
can be used with .alpha.-thio-guanosine nucleotides according to
the manufacturer's instructions to create a phosphorothioate
linkage in the 5'-ppp-5' cap. Additional modified guanosine
nucleotides can be used such as .alpha.-methyl-phosphonate and
seleno-phosphate nucleotides.
[0397] In other embodiments, a long chain of adenine nucleotides
(poly-A tail) can be added to an oligonucleotide in order to
increase stability. The poly-A polymerase can add a chain of
adenine nucleotides to the 3'-end of the oligonucleotide. The
process, called polyadenylation, adds a poly-A tail that can be
between, for example, approximately 80 to approximately 250
residues long, including approximately 80, 90, 100, 110, 120, 130,
140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250
residues long.
[0398] Non-limiting examples of dinucleotides and oligonucleotides
that can be prepared by the methods of the present disclosure are
presented in Table 5.
TABLE-US-00005 TABLE 5 5-1 ##STR00318##
O-((2R,3S,5R)-5-(6-amino-9H-purin-9-yl)-2- (((tert-
butyldiphenylsilyl)oxy)methyl)tetrahydrofuran- 3-yl)
O-(((2R,3S,5R)-5-(6-benzamido-9H- purin-9-yl)-3-((tert-
butyldiphenylsilyl)oxy)tetrahydrofuran-2- yl)methyl) O-hydrogen
(R)-phosphorothioate 5-2 ##STR00319##
O-((2R,3S,5R)-5-(6-amino-9H-purin-9-yl)-2- (((tert-
butyldiphenylsilyl)oxy)methyl)tetrahydrofuran- 3-yl)
O-(((2R,3S,5R)-5-(4-benzamido-2- oxopyrimidin-1(2H)-yl)-3-((tert-
butyldiphenylsilyl)oxy)tetrahydrofuran-2- yl)methyl) O-hydrogen
(S)-phosphorothioate 5-3 ##STR00320##
O-((2R,3S,5R)-5-(6-amino-9H-purin-9-yl)-2- (((tert-
butyldiphenylsilyl)oxy)methyl)tetrahydrofuran- 3-yl)
O-(((2R,3S,5R)-3-((tert-
butyldiphenylsilyl)oxy)-5-(5-methyl-2,4-dioxo-
3,4-dihydropyrimidin-1(2H)- yl)tetrahydrofuran-2-yl)methy
O-hydrogen (S)-phosphorothioate 5-4 ##STR00321##
O-((2R,3S,5R)-5-(6-amino-9H-purin-9-yl)-2- (((tert-
butyldiphenylsilyl)oxy)methyl)tetrahydrofuran- 3-yl)
O-(((2R,3S,5R)-3-((tert-
butyldiphenylsilyl)oxy)-5-(2-(isobutylamino)-
6-oxo-1,6-dihydro-9H-purin-9- yl)tetrahydrofuran-2-yl)methyl)
O-hydrogen (S)-phosphorothioate 5-5 ##STR00322##
O-((2R,3S,5R)-5-(4-amino-2-oxopyrimidin- 1(2H)-yl)-2-(((tert-
butyldiphenylsilyl)oxy)methyl)tetrahydrofuran- 3-yl)
O-(((2R,3S,5R)-5-(4-benzamido-2- oxopyrimidin-1(2H)-yl)-3-((tert-
butyldiphenylsilyl)oxy)tetrahydrofuran-2- yl)methyl) O-hydrogen
(R)-phosphorothioate 5-6 ##STR00323##
O-((2R,3S,5R)-5-(4-amino-2-oxopyrimidin- 1(2H)-yl)-2-(((tert-
butyldiphenylsilyl)oxy)methyl)tetrahydrofuran- 3-yl)
O-(((2R,3S,5R)-3-((tert-
butyldiphenylsilyl)oxy)-5-(5-methyl-2,4-dioxo-
3,4-dihydropyrimidin-1(2H)- yl)tetrahydrofuran-2-yl)methyl)
O-hydrogen (S)-phosphorothioate 5-7 ##STR00324##
O-((2R,3S,5R)-5-(4-amino-2-oxopyrimidin- 1(2H)-yl)-2-(((tert-
butyldiphenylsilyl)oxy)methyl)tetrahydrofuran- 3-yl)
O-(((2R,3S,5R)-3-((tert-
butyldiphenylsilyl)oxy)-5-(2-(isobutylamino)-
6-oxo-1,6-dihydro-9H-purin-9- yl)tetrahydrofuran-2-yl)methyl)
O-hydrogen (R)-phosphorothioate 5-8 ##STR00325##
O-(((2R,3S,5R)-3-((tert-
butyldiphenylsilyl)oxy)-5-(5-methyl-2,4-dioxo-
3,4-dihydropyrimidin-1(2H)- yl)tetrahydrofuran-2-yl)methyl)
O-((2R,3S,5R)- 2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-(5-
methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)tetrahydrofuran-3-yl) O-hydrogen (S)- phosphorothioate 5-9
##STR00326## O-(((2R,3S,5R)-3-((tert-
butyldiphenylsilyl)oxy)-5-(2-(isobutylamino)-
6-oxo-1,6-dihydro-9H-purin-9- yl)tetrahydrofuran-2-yl)methyl)
O-((2R,3S,5R)- 2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-(5-
methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)tetrahydrofuran-3-yl) O-hydrogen (S)- phosphorothioate 5-10
##STR00327## O-(((2R,3S,5R)-3-((tert-
butyldiphenylsilyl)oxy)-5-(2-(isobutylamino)-
6-oxo-1,6-dihydro-9H-purin-9- yl)tetrahydrofuran-2-yl)methyl)
O-((2R,3S,5R)- 2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-(2-
formamido-6-oxo-1,6-dihydro-9H-purin-9- yl)tetrahydrofuran-3-yl)
O-hydrogen (R)- phosphorothioate 5-11 ##STR00328##
((2S,3S,5R)-3-azido-5-(5-methyl-2,4-dioxo-
3,4-dihydropyrimidin-1(2H)- yl)tetrahydrofuran-2-yl)methyl
((2R,3S,5R)-2- (((tert-butyldiphenylsilyl)oxy)methyl)-5-(5-
methyl-2,4-dioxo-3,4-dibydropyrimidin-1(2H)-
yl)tetrahydrofuran-3-yl) hydrogen phosphate 5-12 ##STR00329##
((2S,3S,5R)-3-azido-5-(5-methyl-2,4-dioxo-
3,4-dihydropyrimidin-1(2H)- yl)tetrahydrofuran-2-yl)methyl
((2R,3S,5R)-2- ((bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-5-(5-
methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)tetrahydrofuran-3-yl) hydrogen phosphate 5-13 ##STR00330##
O-(((3aR,4R,6R,6aR)-6-(6-benzamido-9H-
purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-
d][1,3]dioxol-4-yl)methyl) O-((2R,3S,5R)-2-
(((tert-butyldimethylsilyl)oxy)methyl)-5-(5-
methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)tetrahydrofuran-3-yl) S-hydrogen phosphorodithioate 5-14
##STR00331## ((2R,3S,5R)-3-((tert-butyldiphenylsilyl)oxy)-5-
(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)tetrahydrofuran-2-yl)methyl ((2R,3S,5R)-2-(((tert-
butyldiphenylsilyl)oxy)methyl)-5-(5-methyl-
2,4-dioxo-3,4-dihydropyrimidin-1(2H)- yl)tetrahydrofuran-3-yl)
hydrogen phosphate 5-15 ##STR00332##
O-((2R,3R,4R,5R)-2-((((((2R,3S,4R,5R)-2-
((((((2R,3R,4R,5R)-5-(6-amino-7,8-dihydro-
9H-purin-9-yl)-2-((((((2R,3S,4R,5R)-5-(6-
amino-9H-purin-9-yl)-4-fluoro-2- (hydroxymethyl)tetrahydrofuran-3-
yl)oxy)sulfidophosphoryl)oxy)methyl)-4- fluorotetrahydrofuran-3-
yl)oxy)sulfidophosphoryl)oxy)methyl)-5-(6- amino-9H-purin-9-yl)-4-
fluorotetrahydrofuran-3- yl)oxy)sulfidophosphoryl)oxy)methyl)-5-(6-
amino-9H-purin-9-yl)-4- fluorotetrahydrofuran-3-yl) O-(((2R,3S,5R)-
3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)tetrahydrofuran- 2-yl)methyl)
phosphorothioate 5-16 ##STR00333##
O-((2R,3S,5R)-2-((((((2R,3R,5R)-2-
((((((2R,3S,5R)-5-(4-amino-2-oxopyrimidin-
1(2H)-yl)-2-((((((2R,3R,5R)-5-(2-amino-6-
oxo-1,6-dihydro-9H-purin-9-yl)-2- (hydroxymethyl)tetrahydrofuran-3-
yl)oxy)sulfidophosphoryl)oxy)methyl)tetrahydrofuran-
3-yl)oxy)sulfidophosphoryl)oxy)methyl)-5-(6-
amino-9H-purin-9-yl)tetrahydrofuran-3-
yl)oxy)sulfidophosphoryl)oxy)methyl)-5-(5-
methyl-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)tetrahydrofuran-3-yl) O-
(((2R,3S,5R)-3-hydroxy-5-(5-methyl-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)- yl)tetrahydrofuran-2-yl)methyl)
phosphorothioate 5-17 Structure see Example 13(a) P(V)-DMT-T16 (all
R) 5-18 Structure see Example 13(b) P(V)-T16 (all R) 5-19 Structure
see Example 13(c) 17-mer TAGTCGACTTGGCCAAT
[0399] In one embodiment, dinucleotides are the compound selected
from group consisting of compounds 5-13, 5-15, and 5-16. In another
embodiment, oligonucleotides are the compound selected from group
consisting of compounds 5-17 and 5-18. In another embodiment,
dinucleotides are the compound selected from group consisting of
compounds 5-1 to 5-10 and compound 5-14. In another embodiment,
dinucleotides and oligonucleotides are the compound selected from
group consisting of compounds 5-11, 5-12, and 5-19.
[0400] Non-limiting examples of CDNs that can be prepared by the
methods of the present disclosure are presented in Table 6.
TABLE-US-00006 TABLE 6 Cpd. No. Structure 6-1 ##STR00334## 6-2
##STR00335## 6-3 ##STR00336## 6-4 ##STR00337## 6-5 ##STR00338## 6-6
##STR00339## 6-7 ##STR00340## 6-8 ##STR00341## 6-9 ##STR00342##
6-10 ##STR00343## 6-11 ##STR00344## 6-12 ##STR00345## 6-13
##STR00346## 6-14 ##STR00347## 6-15 ##STR00348## 6-16 ##STR00349##
6-17 ##STR00350## 6-18 ##STR00351## 6-19 ##STR00352## 6-20
##STR00353## 6-21 ##STR00354## 6-22 ##STR00355## 6-23
##STR00356##
[0401] For compounds of Table 6, when X is O, all structures are
(R/S-P) combinations; and when X is S, all (R/S-P)
combinations.
Methods of Making Peptide/Protein--Nucleic Acid Conjugates
[0402] In another aspect, the present disclosure provides a method
for making peptide/protein--nucleic acid (e.g., nucleosides,
nucleotides, oligonucleotides, polynucleotides) conjugates
comprising P(V)-based linkages. In some embodiments, the linkages
are achiral. In some embodiments, the linkages are stereo-defined
thiophosphate linkages. In some embodiments, the method comprises
(a) reacting a compound of any of the Formulae (I)-(IIIe) with a
nucleophile and (b) reacting the compound formed in step (a) with
another nucleophile, thereby forming a stereo-defined linkage. In
one embodiment, one of the nucleophiles is supplied by nucleic
acids (e.g., 3'-hydroxy and 5'-hydroxy), and another nucleophile is
supplies by a peptide or a protein (e.g., --NH.sub.2, --OH, --SH,
--C(O)NH.sub.2, --C(O)OH, etc.).
[0403] In some embodiments, both reactions are conducted in the
presence of a base. Suitable bases are described above for
preparing oligonucleotides comprising stereo-defined thiophosphate
linkages. Reaction conditions for such reactions are also described
above. A skilled artisan will easily be able to vary the reaction
conditions, such as time, temperature, amounts of the reagents,
etc., to achieve desirable yields.
[0404] In one embodiment, the peptide is about 50 amino acids long,
about 40 amino acids long, about 30 amino acids long, about 20
amino acids long, about 10 amino acids long, about 5 amino acids
long, about 2 amino acids long, or about 1 amino acid long. In some
embodiments, the peptide comprises only naturally-occurring amino
acids. In other embodiments, the peptide comprises naturally- and
non-naturally-occurring amino acids.
[0405] In one embodiment, the nucleic acid is a nucleoside
(naturally-occurring or modified). In another embodiment, the
nucleic acid is a nucleotide (naturally-occurring or modified). In
one embodiment, the nucleic acid is an oligonucleotide comprising
only naturally-occurring nucleosides. In one embodiment, the
nucleic acid is an oligonucleotide comprising naturally-occurring
and modified nucleosides. In another embodiment, the nucleic acid
is an oligonucleotide comprising only modified nucleosides.
Suitable naturally-occurring and modified nucleobases and sugars
are described above. It will be within a purview of a skilled
artisan to select appropriate nucleosides, based on the eventual
application of the conjugates.
[0406] Examples of suitable proteins include, but are not limited
to, enzymes, antibodies, cytokines, hormones, trans-membrane
proteins, etc. More specific examples of proteins that can be used
include, but are not limited to, polyclonal and monoclonal
antibodies, including but are not limited to fully human
antibodies; single chain antibodies; fragments of antibodies;
chimeric antibodies and antigen-binding fragments thereof; domain
antibodies and antigen-binding fragments thereof; interferons
(e.g., alpha, beta, gamma); lymphokines (IL-2, IL-3, IL-4, IL-6);
protein hormones, such as insulin; and enzymes.
[0407] In some embodiments, the methods of making
peptide/protein--nucleic acid comprise a purification step.
Purification can be performed by methods known in the arts such as,
but not limited to, HPLC based purification methods such as, but
not limited to, strong anion exchange HPLC, weak anion exchange
HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction
HPLC (HIC-HPLC).
[0408] In some embodiments, the nucleosides and/or amino acids can
bear a protecting group. Suitable protecting groups are described
above. In some embodiments, the protecting group can be removed by
the methods described above.
[0409] Non-limiting examples of conjugates that can be prepared by
the methods of the present disclosure are presented in Table 7.
TABLE-US-00007 TABLE 7 Cpd. No. Structure 7-1 ##STR00357## 7-2
##STR00358## 7-3 ##STR00359## 7-4 ##STR00360## 7-5 ##STR00361## 7-6
##STR00362## 7-7 ##STR00363## 7-8 ##STR00364## 7-9 ##STR00365##
7-10 ##STR00366## 7-11 ##STR00367## 7-12 ##STR00368## 7-13
##STR00369## 7-14 ##STR00370##
Control of Chirality at the Phosphorous Atom
[0410] The methods described herein are useful for controlling the
configuration of each phosphorus atom in an organosphosphorous (V)
compound (for example, an internucleoside linkage). The novel
reagents described herein permit the specific control of the
chirality at the phosphorus atom. For example, in embodiments
pertaining to oligonucleotides containing thiophosphate
internucleoside linkages, either a Rp or Sp configuration can be
selected in each synthesis cycle, permitting control of the overall
three dimensional structure of the nucleic acid product. In some
embodiments, the selection of Rp or Sp configurations is made to
confer a specific three dimensional superstructure to the nucleic
acid chain.
[0411] In some embodiments, each phosphorous atom can have a Rp
configuration. In other embodiments, each phosphorous atom can have
a Sp configuration. In another embodiment, each phosphorous atom
independently can have a Rp configuration or a Sp configuration. In
another embodiment, the phosphorous atoms alternate between Rp and
Sp such as Rp, Sp, Rp or Sp, Rp, Sp throughout the nucleic acid. In
other specific embodiments, the phosphorous atoms contain repeated
configurations of Rp, Rp, Sp, Sp throughout the nucleic acid. In
yet other embodiments, the nucleic acid comprises all Rp
configurations. In further embodiments, the nucleic acid comprises
all Sp moieties. In some embodiments, the 5' and 3' terminal
internucleoside linkages are of the Sp configuration and the
internal internucleoside linkages are all of the Rp configuration.
The embodiments described herein serve as examples of how the
configuration can be controlled using these methods. The nucleic
acids (oligonucleotides and CDNs) described herein are not limited
to these configuration patterns. It will be evident to one skilled
in the art that other variations and alternations in the Rp and Sp
configurations are possible and depend on the use and applications
of the nucleic acid.
Purity Determination of Phosphorous Configurations
[0412] The purity of the configuration at each phosphorous atom in
a organosphosphorous (V) compound (e.g., an oligonucleotide, a CDN,
a peptide-oligonucleotide conjugate, etc.) is determined using
conventional analytical methods such as, but not limited to,
.sup.31P NMR spectroscopy or reverse-phase HPLC. Using methods
described herein, in an embodiment, each phosphorous atom of a
organosphosphorous (V) compound can be more than 80%
diastereomerically pure. In an embodiment, each phosphorous atom of
a organosphosphorous (V) compound can be more than 60%
diastereomerically pure. In an embodiment, each phosphorous atom of
a organosphosphorous (V) compound can be more than 70%
diastereomerically pure. In an embodiment, each phosphorous atom of
a organosphosphorous (V) compound can be more than 85%
diastereomerically pure. In an embodiment, each phosphorous atom of
a organosphosphorous (V) compound can be more than 90%
diastereomerically pure. In an embodiment, each phosphorous atom of
a organosphosphorous (V) compound can be more than 95%
diastereomerically pure. In another embodiment, each phosphorous
atom of a organosphosphorous (V) compound can be more than 98%
diastereomerically pure. In another embodiment, each phosphorous
atom of a organosphosphorous (V) compound can be more than 99%
diastereomerically pure. In an embodiment, each phosphorous atom of
a organosphosphorous (V) compound can be more than about 60%, more
than about 70%, more than about 80%, more than about 83%, more than
about 84%, more than about 85%, more than about 86%, more than
about 87%, more than about 88%, more than about 89%, more than
about 90%, more than about 91%, more than about 92%, more than
about 93%, more than about 94%, more than about 95%, more than
about 96%, more than about 97%, more than about 98%, or more than
about 99% diastereomerically pure. In one embodiment, each
phosphorous atom of a organosphosphorous (V) compound can be from
about 60% to about 99.9% diastereomerically pure. In one
embodiment, each phosphorous atom of a organosphosphorous (V)
compound can be from about 60% to about 99% diastereomerically
pure. In one embodiment, each phosphorous atom of a
organosphosphorous (V) compound can be from about 60% to about 70%
diastereomerically pure. In one embodiment, each phosphorous atom
of a organosphosphorous (V) compound can be from about 70% to about
80% diastereomerically pure. In one embodiment, each phosphorous
atom of a organosphosphorous (V) compound can be from about 80% to
about 90% diastereomerically pure. In one embodiment, each
phosphorous atom of a organosphosphorous (V) compound can be from
about 80% to about 99% diastereomerically pure. In one embodiment,
each phosphorous atom of a organosphosphorous (V) compound can be
from about 85% to about 95% diastereomerically pure. In one
embodiment, each phosphorous atom of a organosphosphorous (V)
compound can be from about 90% to about 95% diastereomerically
pure. In one embodiment, each phosphorous atom of a nucleic acid
can be from about 95% to about 99% diastereomerically pure. In one
embodiment, each phosphorous atom of a organosphosphorous (V)
compound can be from about 90% to about 99.9% diastereomerically
pure.
[0413] The amount of a particular configuration over another
configuration affects the three-dimensional structure of a
organosphosphorous (V) compound, such as nucleic acids, as well as
their stability. Accordingly, different configurations affect the
biological, chemical, and physical properties of the nucleic acids.
In one embodiment, the nucleic acid comprises a greater percentage
of Sp configuration than Rp configuration. In another embodiment,
the nucleic acid comprises a greater percentage of Rp configuration
than Sp configuration. In another embodiment, the nucleic acid
comprises the same percentage of Rp configuration as Sp
configuration. In one embodiment, the nucleic acid can comprise
0-20% Rp configuration. In one embodiment, the nucleic acid can
comprise 20-40% Rp configuration. In one embodiment, the nucleic
acid can comprise 40-60% Rp configuration. In one embodiment, the
nucleic acid can comprise 60-80% Rp configuration. In one
embodiment, the nucleic acid can comprise 80-100% Re configuration.
In one embodiment, the nucleic acid can comprise 0-20% Rp
configuration. In one embodiment, the nucleic acid can comprise
20-40% Sp configuration. In one embodiment, the nucleic acid can
comprise 40-60% Sp configuration. In one embodiment, the nucleic
acid can comprise 60-80% Sp configuration. In one embodiment, the
nucleic acid can comprise 80-100% Sp configuration.
EXAMPLES
[0414] The invention is further defined in the following Examples.
It should be understood that the Examples are given by way of
illustration only. From the above discussion and the Examples, one
skilled in the art can ascertain the essential characteristics of
the invention, and without departing from the spirit and scope
thereof, can make various changes and modifications to adapt the
invention to various uses and conditions. As a result, the
invention is not limited by the illustrative examples set forth
hereinbelow, but rather is defined by the claims appended
hereto.
Example 1(a)
Preparation of the Limonene-P(V) Reagents
##STR00371##
[0415] Preparation of Thio-Phosphoric Acid Triethylamine.
[0416] Step (1): To a mixture of phosphorus pentasulfide (30.0 g,
132 mmol, 98 mass %) in toluene (240 mL, 8 mL/g) was charged
pentafluorothiophenol (PFTP) (55 g, 266.58 mmol, 97 mass %) at
21.degree. C. The batch was inert by flushing with N.sub.2 for 2
min. Triethylamine (TEA) (39 mL, 277 mmol, 99 mass %) was added
over a period of 0.5 hours. The batch temperature went to
45.degree. C. at the end of the addition. At the end of TEA
addition, the batch became a nearly clear yellow solution and then
gradually turned cloudy in 0.5 hours. The batch temperature slowly
dropped to ambient temperature over 0.5 hours (by air cooling).
HPLC analysis of a sample at 3 hours indicated that relative area
percent ("RAP") of PFTP vs. product was less than 5%.
[0417] Step (2): The mixture was stirred at ambient temperature
overnight. The slurry was then filtered, and the reactor was rinsed
with toluene (30 mL.times.2) and the rinses were applied to the
cake washes. The filtration was rapid, and HPLC analysis of the
cake indicated very little loss of product.
[0418] Steps (3) and (4): The combined filtrates were concentrated
under vacuum to 105 g (.about.3.5 v). Methanol (180 mL, 6 v) was
added, followed by heptane (180 mL, 6 v). The biphasic mixture was
stirred for 15 min. Water (150 mL, 5v) was added over 30 min. After
35 mL of water was added, seed (0.3 g, 1%) was added. After the
seed was added, a slurry was formed within a minute.
[0419] Step (5): After the addition of water was complete, the
batch was mixed for 1 h. The batch was filtered. The reactor was
rinsed with a mixture of 3:2 water/methanol (75 mL), and the rinse
was applied for the cake wash. The filter cake was washed with
water (90 mL.times.2), then heptane (45 mL.times.2). The cake was
dried in vacuo for 15 h at 50.degree. C. 73 g of thio-phosphoric
acid TEA salt was collected as a white solid (93% as-is yield). The
product was characterized by .sup.1H NMR (500 MHz, chloroform-d)
.delta. 8.99-8.60 (m, 1H), 3.40-3.20 (m, 6H), 1.51-1.36 (m, 9H);
and P NMR .delta. 99.35. Process flow diagram for preparing
thio-phosphoric acid TEA salt is shown below:
##STR00372##
Preparation of the Limonene P(V) Reagent A (Compound 1-31) from
Trans-Limonene.
[0420] Step (1): A solution of
bis[(2,3,4,5,6-pentafluorophenyl)sulfanyl]-sulfido-thioxophosphane
triethylammonium (1.00 g, 1.68 mmol, 100 mass %) and
(1R,3R,6S)-3-isopropenyl-6-methyl-7-oxabicyclo[4.1.0]heptane (0.383
g, 2.52 mmol, 100 mass %) in dichloromethane (DCM) (5.0 mL, 5 v)
was made inert by flushing with nitrogen. Trifluoroacetic acid
(0.19 mL, 2.52 mmol, 100 mass %) was added at 21.degree. C. The
mixture was then heated at 35.degree. C. for one hour. HPLC
analysis of a sample indicated that RAP of P-SH SM vs. product was
less than 3%.
[0421] Steps (2) and (3): The reaction mixture was cooled to
ambient temperature, and hexanes (10 mL, 10 v) was added into the
batch (a biphasic mixture). The resulting stream was washed with
water (5 mL), saturated NaHCO.sub.3 (10 mL), and 10%
KH.sub.2PO.sub.4 (3 mL). The organic phase was filtered through a
MgSO.sub.4 pad, and concentrated to .about.3 mL. Methanol (5 mL)
was added, the batch was concentrated to .about.3 mL. Addition of
methanol (5 mL) was repeated and the reaction mixture was
concentrated to .about.3 mL. The mixture was cooled to 5-10.degree.
C. and stirred for 5 min. The resulting slurry was filtered, and
the reactor and cake were washed with cold methanol (1 mL). The
cake was dried, and weighed 0.56 g in 75% as-is yield. The product
was characterized by .sup.1H NMR (500 MHz, chloroform-d) .delta.
5.09-4.95 (m, 1H), 4.81-4.66 (m, 1H), 3.07-2.89 (m, 1H), 2.44 (br
s, 1H), 2.18-2.09 (m, 2H), 2.09-2.03 (m, 1H), 1.80-1.70 (m, 2H),
1.69-1.67 (m, 6H), 1.61-1.53 (m, 3H); and P NMR .delta. 101.61.
Process flow diagram for preparing limonene P(V) reagent A is shown
below:
##STR00373##
Preparation of the Limonene P(V) Reagent B (Compound 1-33) from
Cis-Limonene.
[0422] The preparation of limonene P(V) B used the same procedure
of making limonene P(V) A except that tran-limonene was replaced by
cis-limonene. Process flow diagram for preparing limonene P(V)
reagent B is shown below:
##STR00374##
Example 1(b)
Alternative Preparation of Limonene P(V) Reagent B (Compound 1-33)
from Cis-Limonene
##STR00375##
[0424] Step (1): The thio-phosphoric TEA salt was prepared
according to the procedure described in paragraphs [361]-[364].
[0425] To a solution of thio-phosphoric acid TEA salt (232 g, 389.6
mmol, 100 mass %) and cis-(+)-limonene oxide (90 g, 591.21 mmol,
100 mass %) in chloroform (2.5 L, contains 0.5-1% EtOH as a
stabilizer) was added dibutyl phosphate (DBP) (83 mL, 439.1 mmol,
100 mass %) at room temperature under nitrogen. Then,
dichloroacetic acid (72 mL, 872.2 mmol, 100 mass %) was added
immediately, with rinse of chloroform (50 mL). After the addition
of the acids, the batch temp increased to 26.degree. C. The
resulting mixture was warmed to 55-60.degree. C. over about 40 min.
After 2.5 hours, the reaction gave two diastereomers with a ratio
of about 8:1. The HPLC retention time for these two diastereomers
is 1.932 min and 1.993 min respectively.
[0426] Step (2): The batch from step (1) was concentrated to about
1.3 L at 35-60.degree. C. under vacuum (200 torr) over .about.2
hours. To this mixture, hexane was added (2 L). The resulting batch
was quickly washed with 10% aqueous K.sub.2HPO.sub.4 (1.5 L). The
batch was agitated for less than 5 min and allowed for phase
separation. After separation, the organic layer was washed with 10%
aqueous KH.sub.2PO.sub.4 (0.5 L), then water (0.5 L) to remove
PFTP, DCA, DBP, and NEt.sub.3. Then the organic layer was
concentrated to 0.6 L at 30-60.degree. C. under vacuum (200 torr)
over 2 hours.
[0427] Step (3): To the batch from step (2) MeOH (0.75 L) was added
and the batch was concentrated to about 0.5 L at 30-60.degree. C.
under vacuum. Then MeOH (1.0 L) was added and the batch was
concentrated to about 1.5 L at 30-60.degree. C. under vacuum. The
resulting slurry was heated to about 60.degree. C. and stirred
until all solids were dissolved. The batch was then cooled to
20.degree. C. over a period of 1 hour. Water (100 mL) was added at
30-40.degree. C. during the cooling period. The batch was stirred
at 20.degree. C. for 15-24 h. The resulting slurry was filtered.
The filtrate was recycled and the new slurry was transferred into
the filter. The filter cake was washed with 10% water in MeOH (100
mL) to afford two diastereomers with a ratio of greater than 20:1
(in this experiment, the diasteromer ratio (d.r.) is 98:2). If the
ratio is less than 20:1, then the cake is re-dissolved in MeOH (10
L/kg of cake) at 60.degree. C., followed by cooling to 20.degree.
C., and water (0.7 L/kg of cake) is added and mixed with the batch
for 15-24 h. Then, the filtration gives a cake with the desired
purity.
[0428] The cake (125 g) was then dissolved in DCM (0.3 L). The
batch was solvent-swapped to heptane, and distilled to 0.5 L. After
stirring for 1 h at 20.degree. C., the slurry was filtered. The
filtrate was recycled and the new slurry was transferred into the
filter. The filter cake was washed with heptane (50 mL.times.2) and
dried under vacuum at 50.degree. C. to afford the desired product
(105 g, yield 59%, d.r.>99:1; ee>99:1.) The chiral HPLC
retention time of two enantiomers is 7.743 min and 8.199 min
respectively. .sup.1H NMR (500 MHz, chloroform-d) .delta. 5.09-4.95
(m, 1H), 4.81-4.66 (m, 1H), 3.07-2.89 (m, 1H), 2.44 (br s, 1H),
2.18-2.09 (m, 2H), 2.09-2.03 (m, 1H), 1.80-1.70 (m, 2H), 1.69-1.67
(m, 6H), 1.61-1.53 (m, 3H); and P NMR .delta. 101.61.
[0429] The process flow diagram for preparing limonene P(V) reagent
B according to Example 1(b) is shown below:
##STR00376##
Example 2
Formation of a Stereo-Defined Phosphorothioate Internucleoside
Linkage Using the Limonene P(V) Reagent
[0430] The limonene P(V) reagent from Example 1 was used to
stereoselectively form a phosphorothioate internucleoside
linkage.
[0431] (a) A deoxythymidine nucleoside bearing a
5'-di-(p-methoxyphenyl)phenylmethyl ether 4',4'-dimethoxytrityl
(DMTr) protecting group was reacted with either the (+) Sp or the
(-) Rp stereoisomer of the limonene P(V) reagent (see Examples 1(a)
and 1(b) in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) at 25.degree. C. for 10 min.
[0432] (b) To the reaction mixture, a 3'-tert-butyldimethylsilyl
ether (TBS) of deoxythymidine nucleoside was added in the presence
of acetonitrile (MeCN), tetrahydrofuran (THF) and
tetra-n-butylammonium fluoride (TBAF). The reaction was allowed to
proceed at 25.degree. C. for several hours, yielding a dinucleotide
containing a phosphorothioate internucleoside linkage. HPLC
analysis showed that (S)-limonene P(V) gives (S)-nucleoside dimer,
and (R)-limonene P(V) gives (R)-nucleoside dimer, with the overall
retention of stereochemistry in both cases.
Example 3
Preparation of P(S).sub.2 P(V) Reagents
Method A
[0433] To a solution of O,O-bis(4-nitrophenyl) S-hydrogen
phosphorodithioate triethylammonium salt (4.20 mmol) in
acetonitrile (0.10 M) was added dibutylphosphate (16.8 mmol) and
ethylene sulfide (16.8 mmol). The resulting solution was heated at
80.degree. C. for 18 hours. Upon cooling to ambient temperature,
the solvent was removed in vacuo and the resulting residue applied
to silica gel. The product was eluted with dichloromethane/hexane
(3:1). The solid obtained was dissolved in dichloromethane and
precipitated from hexane affording the title compound (3.19 mmol,
76% yield) as a white solid. The reaction can be represented by the
following scheme:
##STR00377##
ii. Method B (General Scheme)
##STR00378##
[0434] Dithiol (1.00 eq) was added to PCl.sub.3 (3.00 eq) at room
temperature. The mixture was stirred for 3 hours (HCl bubbles were
observed during this reaction). The excess PCl.sub.3 was distilled
out by a simple distillation and the compound was stored under
vacuum giving Intermediate 1.
[0435] Intermediate 1 (1.0 eq) in deoxygenized MeCN (0.1 M) was
added dropwise to a stirred solution of Leaving Group-H (LG-H, 1.0
eq.) and DIEA (1.10 eq) in dry, degassed MeCN (73 mL) at 0.degree.
C. After stirring for 1 h at the same temperature, the reaction
mixture was warmed to room temperature and stirred for an
additional 2 h. The P(III) intermediate was then sulfurized with
either S.sub.8(3 eq.) in CS.sub.2 (20 eq.) or EDITH (1 eq.). The
P(V) Reagents were obtained by crystallization from alcoholic
solvents (IPA, MeOH, nBuOH, EtOH, etc.)
2-(4-nitrophenoxy)-1,3,2-dithiaphospholane 2-sulfide (Compound
2-1)
##STR00379##
[0436] Illustration 1
##STR00380##
[0437] Step 1. Synthesis of 2-chloro-1,3,2-dithiaphospholane
(2-ii)
[0438] 32.0 mmol of phosphorus (III) chloride and 1 equiv. of
ethane-1,2-dithiol were mixed at room temperature and stirred for 3
hours. Following completion of the reaction, the mixture was
distilled to afford 2-chloro-1,3,2-dithiaphospholane (5.08 g, 32.0
mmol, quantitative yield).
Step 2. Synthesis of 2-(4-nitrophenoxy)-1,3,2-dithiaphospholane
2-sulfide (Compound 2-1)
[0439] To a solution of 32.0 mmol of
2-chloro-1,3,2-dithiaphospholane and 1 equiv. of 4-nitrophenol in
MeCN [0.1 M] was added 1.0 equiv. of DIPEA at room temperature. The
mixture was stirred for 3 h, followed by a solution of S.sub.8 in
CS.sub.2 (8.0 equiv., 24 wt %). The resulting mixture was stirred
for 3 h. The crude product was recrystallized from 2-propanol (ca.
5 mL/g of expected product) to afford
2-(4-nitrophenoxy)-1,3,2-dithiaphospholane 2-sulfide (6.0 g, 20.4
mmol, 65%).
Illustration 2
##STR00381##
[0440] Synthesis of 2-chloro-1,3,2-dithiaphospholane (2-ii)
[0441] 1,2-Ethanedithiol (1.00 eq., 27.34 g, 24.35 mL, 290.2 mmol)
was added (5 min) to PCl.sub.3 (3.00 eq., 119.57 g, 75.97 mL,
870.70 mmol) at room temperature. The mixture was stirred for 3 h
(HCl bubbles were observed during this reaction). The PCl.sub.3
excess was distilled out by a simple distillation and the compound
was stored under vacuum. The compound was obtained as transparent
liquid in quantitative yield (45.8 g). .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 3.82-3.65 (m, 2H), 3.65-3.48 (m, 2H). .sup.13C
NMR (150 MHz, CDCl.sub.3) .delta. 42.82, 42.80. .sup.31P NMR (244
MHz, CDCl.sub.3) .delta. 167.92.
Synthesis of
2-(4-Nitrophenylthiol)-1,3,2-dithiaphospholane-2-sulfide (Compound
2-1)
[0442] 2-Chloro-1,3,2-dithiophospholane (1.0 eq., 22.9 g, 145.01
mmol) in deoxygenized MeCN (229.5 mL) was added dropwise to a
stirred solution of 4-Nitrophenol (1.0 eq., 20.17 g, 145.01 mmol)
and DIEA (1.10 eq., 20.6 g, 27.8 mL, 159.5 mmol) in dry, degassed
MeCN (73 mL) at -40.degree. C. After stirring for 1 h at the same
temperature, the reaction mixture was warmed to room temperature
and stirred for an additional 2 h 30 min. The mixture was analyzed
by NMR (.sup.31P and .sup.1H). The intermediate
2-(4-nitrophenoxy)-1,3,2-dithiaphospholane (3a2) was stable during
this process. .sup.1H NMR (400 MHz, CDCl3) .delta. 8.03 (d, J=9.2
Hz, 2H), 7.03 (d, J=9.2 Hz, 2H), 3.17 (m, 4H). .sup.31P NMR (244
MHz, CDCl3) .delta. 160.56.
[0443] A solution of S.sub.8 (3.0 eq., 13.95 g, 435.03 mmol)
dissolved in CS.sub.2 (20 eq., 175 mL) and was slowly added to the
mixture above for 20 min., at room temperature and the resultant
light yellow heterogeneous mixture was vigorously stirred overnight
at 35.degree. C.
[0444] Purification and Recrystallization: IPA (250 mL) was added
at room temperature and then was warmed until complete solution.
Then it was cooled to room temperature and was stirred 3 hours. The
precipitated solid was filtrated and washed with additional IPA (50
mL). The compound was obtained as white solid (30.0 g, 70.6%, this
compound has an impurity in 7% but It can be used in the next
step). The solid was solved in DCM (50 mL) and was added silica gel
(10 g) and it was dried under reduced pressure. After, this solid
was loaded onto a silica gel column and the mixture was washed with
Hexane:DCM 1:1 (600 mL). The filtrate was concentrated under
reduced pressure to dry to afford the desired compound 2-1 as white
solid (22.0 g, 51.8%, Rf.: 0.25 in DCM/Hexane 1:1). .sup.1H NMR
(400 MHz. CDCl3) .delta. 8.26 (d, J=9.0 Hz, 2H), 7.41 (dd, J=9.1,
2.0 Hz, 2H), 3.88-3.66 (m, 4H). .sup.13C NMR (150 MHz, CDCl3)
.delta. 155.67 and 155.58, 145.33 and 145.32, 125.51 and 125.49,
122.78 and 122.75, 42.09. .sup.31P NMR (244 MHz, CDCl3) .delta.
120.04.
[0445] The following compounds 3b-3t were prepared by utilizing
method B.
b. 2-phenoxy-1,3,2-dithiaphospholane 2-sulfide (Compound 2-2)
##STR00382##
[0447] The title compound was prepared using method B and LG-H is
phenol. .sup.1H NMR (600 MHz, Chloroform-d) .delta. 7.40-7.33 (m,
2H), 7.29-7.21 (m, 3H), 3.76-3.66 (m, 2H), 3.64-3.56 (m, 2H).
.sup.13C NMR (151 MHz, Chloroform-d) .delta. 151.13 (d, J=13.2 Hz),
129.65 (d, J=2.4 Hz), 125.93 (d, J=2.6 Hz), 122.16 (d, J=5.0 Hz),
41.95. .sup.31P NMR (202 MHz, Chloroform-d) .delta. 119.02 (p,
J=16.3 Hz). HRMS (ESI-TOF) m/z Calcd for
C.sub.8H.sub.10OPS.sub.3.sup.+ [M+H].sup.+ 248.9626, found
248.9641.
c. 2-(phenylthio)-1,3,2-dithiaphospholane 2-sulfide (Compound
2-3)
##STR00383##
[0449] The title compound was prepared using method B and LG-H is
benzenethiol. .sup.1H NMR (600 MHz, Chloroform-d) .delta. 7.70-7.64
(m, 2H), 7.50-7.45 (m, 1H), 7.44-7.38 (m, 2H), 3.62-3.50 (m, 2H),
3.23-3.12 (m, 2H). .sup.13C NMR (151 MHz, Chloroform-d) .delta.
136.64 (d, J=4.7 Hz), 130.59 (d, J=4.4 Hz), 130.51 (d, J=8.3 Hz),
129.36 (d, J=3.7 Hz), 42.92. .sup.31P NMR (202 MHz, Chloroform-d)
.delta. 108.61. HRMS (ESI-TOF) m/z Calcd for
C.sub.8H.sub.10PS.sub.4.sup.+ [M+H].sup.+ 264.9397, found
264.9423.
d. 2-((4-nitrophenyl)thio)-1,3,2-dithiaphospholane 2-sulfide
(Compound 2-4)
##STR00384##
[0451] The title compound was prepared using method B and LG-H is
4-nitrobenzenethiol. .sup.1H NMR (600 MHz, Chloroform-d) .delta.
8.26 (d, J=8.0 Hz, 2H), 7.83 (dd, J=8.9, 2.4 Hz, 2H), 3.77-3.66 (m,
2H), 3.54-3.44 (m, 2H). .sup.13C NMR (151 MHz, Chloroform-d)
.delta. 149.00 (d, J=4.4 Hz), 138.30 (d, J=7.8 Hz), 136.81 (d,
J=4.6 Hz), 124.19 (d, J=3.0 Hz), 42.85. .sup.31P NMR (202 MHz,
Chloroform-d) .delta. 105.41 (tt, J=19.2, 16.0 Hz). HRMS (ESI-TOF)
m/z Calcd for C.sub.8H.sub.9NO.sub.2PS.sub.4.sup.+ [M+H].sup.+
309.9248, found 309.9240.
e. 2-(4-bromophenoxy)-1,3,2-dithiaphospholane 2-sulfide (Compound
2-5)
##STR00385##
[0453] The title compound was prepared using method B and LG-H is
4-bromophenol. .sup.1H NMR (600 MHz, Chloroform-d) .delta.
7.51-7.45 (m, 2H), 7.15 (d, J=12.0 Hz, 2H), 3.77-3.68 (m, 2H),
3.67-3.59 (m, 2H), 13C NMR (151 MHz, Chloroform-d) .delta. 150.04
(d, J=13.2 Hz), 132.69 (d, J=2.3 Hz), 123.94 (d, J=5.2 Hz), 119.17
(d, J=3.4 Hz), 42.00. 31P NMR (202 MHz, Chloroform-d) .delta.
119.85 (ddd, J=32.4, 18.5, 14.3 Hz). HRMS (ESI-TOF) m/z Calcd for
C.sub.8H.sub.9BrOPS.sub.3.sup.+ [M+H].sup.+ 326.8731, found
326.8758.
f. 2-(4-chlorophenoxy)-1,3,2-dithiaphospholane 2-sulfide (Compound
2-6)
##STR00386##
[0455] The title compound was prepared using method B and LG-H is
4-chlorophenol. .sup.1H NMR (500 MHz, Chloroform-d) .delta. 7.32
(t, J=1.6 Hz, 2H), 7.21 (d, J=2.2 Hz, 2H), 3.79-3.68 (m, 2H),
3.68-3.59 (m, 2H), 13C NMR (151 MHz, Chloroform-d) .delta. 149.51
(d, J=13.2 Hz), 131.48 (d, J=3.3 Hz), 129.73 (d, J=2.2 Hz), 123.53
(d, J=5.2 Hz), 41.99. 31P NMR (202 MHz, Chloroform-d) .delta.
119.94 (ddd, J=32.5, 18.6, 14.4 Hz). HRMS (ESI-TOF) m/z Calcd for
C.sub.8H.sub.9ClOPS.sub.3.sup.+ [M+H].sup.+ 282.9236, found
282.9285.
g. 2-((4-chlorophenyl)thio)-1,3,2-dithiaphospholane 2-sulfide
(Compound 2-7)
##STR00387##
[0457] The title compound was prepared using method B and LG-H is
4-chlorobenzenethiol. .sup.1H NMR (600 MHz, Chloroform-d) .delta.
7.58 (d, J=8.5 Hz, 2H), 7.39 (dd, J=8.6, 0.9 Hz, 2H), 3.68-3.54 (m,
2H), 3.36-3.18 (m, 2H), 13C NMR (151 MHz, Chloroform-d) .delta.
137.69 (d, J=4.8 Hz), 137.31 (d, J=5.2 Hz), 129.65 (d, J=3.8 Hz),
128.94 (d, J=8.2 Hz), 42.92. 31P NMR (202 MHz, Chloroform-d)
.delta. 107.82. HRMS (ESI-TOF) m/z Calcd for
C.sub.8H.sub.9ClPS.sub.4.sup.+ [M+H].sup.+ 298.9008, found
298.9022.
h. 2-(perfluorophenoxy)-1,3,2-dithiaphospholane 2-sulfide (Compound
2-8)
##STR00388##
[0459] The title compound was prepared using method B and LG-H is
pentafluorophenol. .sup.1H NMR (500 MHz, Chloroform-d) .delta.
3.89-3.76 (m, 4H). .sup.13C NMR (151 MHz, Chloroform-d) .delta.
142.05 (m), 139.26 (m), 138.04 (m), 125.94 (d, J=14.8 Hz), 42.41.
.sup.31P NMR (202 MHz, Chloroform-d) .delta. 127.31 (p, J=17.8 Hz).
HRMS (ESI-TOF) m/z Calcd for C.sub.8H.sub.9ClPS.sub.4.sup.+
[M+H].sup.+ 298.9008, found 298.9022.
i. 2-((perfluorophenyl)thio)-1,3,2-dithiaphospholane 2-sulfide
(Compound 2-9)
##STR00389##
[0461] The title compound was prepared using method B and LG-H is
2,3,4,5,6-pentafluorobenzenethiol. .sup.1H NMR (600 MHz,
Chloroform-d) .delta. 3.81-3.63 (m, 4H). .sup.13C NMR (151 MHz,
Chloroform-d) .delta. 148.30 (ddt, J=250.7, 11.1, 4.1 Hz), 143.27
(dddt, J=259.3, 13.6, 9.0, 4.7 Hz), 138.16 (m), 105.78 (m), 43.00.
31P NMR (202 MHz, Chloroform-d) .delta. 103.71. HRMS (ESI-TOF) m/z
Calcd for C.sub.8H.sub.5F.sub.5PS.sub.4.sup.+ [M+H].sup.+ 354.8926,
found 354.8935.
j. 2-(4-methoxyphenoxy)-1,3,2-dithiaphospholane 2-sulfide (Compound
2-10)
##STR00390##
[0463] The title compound was prepared using method B and LG-H is
4-methoxyphenol. .sup.1H NMR (600 MHz, Chloroform-d) .delta.
7.21-7.15 (m, 2H), 6.89-6.83 (m, 2H), 3.80 (s, 3H), 3.73-3.64 (m,
2H), 3.61-3.53 (m, 2H), 13C NMR (126 MHz, Chloroform-d) .delta.
157.43 (d, J=2.9 Hz), 144.64 (d, J=13.4 Hz), 123.09 (d, J=4.8 Hz),
114.56 (d, J=2.8 Hz), 55.71, 41.95. 31P NMR (202 MHz. Chloroform-d)
.delta. 120.24 (ddd, J=32.0, 18.0, 13.7 Hz). HRMS (ESI-TOF) m/z
Calcd for C.sub.9H.sub.12O.sub.2PS.sub.3.sup.+ [M+H].sup.+
278.9732. found 278.9755.
k. 2-((4-methoxyphenyl)thio)-1,3,2-dithiaphospholane 2-sulfide
(Compound 2-11)
##STR00391##
[0465] The title compound was prepared using method B and LG-H is
4-methoxybenzenethiol. .sup.1H NMR (500 MHz, Chloroform-d) .delta.
7.61-7.54 (m, 2H), 6.95-6.89 (m, 2H), 3.84 (s, 3H), 3.61-3.49 (m,
2H), 3.22-3.11 (m, 2H), 13C NMR (151 MHz, Chloroform-d) .delta.
161.67 (d, J=4.1 Hz), 138.22 (d, J=4.4 Hz), 121.36 (d, J=7.9 Hz),
114.89 (d, J=3.5 Hz), 55.58, 42.94. 31P NMR (202 MHz, Chloroform-d)
.delta. 110.45-109.95 (m). HRMS (ESI-TOF) m/z Calcd for
C.sub.9H.sub.12OPS.sub.4.sup.+ [M+H].sup.+ 294.9503, found
294.9527.
l. 2-(4-(trifluoromethyl)phenoxy)-1,3,2-dithiaphospholane 2-sulfide
(Compound 2-12)
##STR00392##
[0467] The title compound was prepared using method B and LG-H is
4-(trifluoromethyl)phenol. .sup.1H NMR (400 MHz, Chloroform-d)
.delta. 7.64 (d, J=8.4 Hz, 2H), 7.42-7.33 (m, 2H), 3.83-3.61 (m,
4H), 13C NMR (151 MHz, Chloroform-d) .delta. 152.85 (d, J=13.0 Hz),
127.51 (qd, J=33.0, 2.7 Hz), 126.45 (p, J=3.6 Hz), 123.36 (d,
J=272.2 Hz), 121.90 (d, J=5.1 Hz), 41.42. 31P NMR (202 MHz,
Chloroform-d) .delta. 119.58 (p, J=16.3 Hz). HRMS (ESI-TOF) m/z
Calcd for C.sub.9H.sub.9F.sub.3OPS.sub.3.sup.+ [M+H].sup.+
316.9500, found 316.9515.
m. 4-((2-sulfido-1,3,2-dithiaphospholan-2-yl)oxy)benzonitrile
(Compound 2-13)
##STR00393##
[0469] The title compound was prepared using method B and LG-H is
4-hydroxybenzonitrile. .sup.1H NMR (600 MHz, Chloroform-d) .delta.
7.75-7.63 (m, 2H), 7.37 (dd, J=8.9, 2.0 Hz, 2H), 3.84-3.63 (m, 4H).
.sup.13C NMR (151 MHz, Chloroform-d) .delta. 154.19 (d, J=12.6 Hz),
133.95 (d, J=2.6 Hz), 123.12 (d, J=5.3 Hz), 118.29 (d, J=1.3 Hz),
109.81 (d, J=2.7 Hz), 42.05. 31P NMR (202 MHz, Chloroform-d)
.delta. 119.80 (p, J=17.3, 16.7 Hz). HRMS (ESI-TOF) m/z Calcd for
C.sub.9H.sub.9NOPS.sub.3.sup.+ [M+H].sup.+ 273.9578, found
273.9587.
n. 2-(4-fluorophenoxy)-1,3,2-dithiaphospholane 2-sulfide (Compound
2-14)
##STR00394##
[0471] The title compound was prepared using method B and LG-H is
4-fluorophenol. .sup.1H NMR (500 MHz, Chloroform-d) .delta.
7.25-7.19 (m, 2H), 7.08-7.01 (m, 2H), 3.78-3.67 (m, 2H), 3.67-3.58
(m, 2H), 13C NMR (151 MHz, Chloroform-d) .delta. 160.38 (dd,
J=244.9, 3.2 Hz), 146.85 (dd, J=13.3, 2.6 Hz), 123.61 (dd, J=8.5,
4.9 Hz), 116.30 (dd, J=23.6, 2.4 Hz), 41.98. 31P NMR (202 MHz,
Chloroform-d) .delta. 120.62-120.08 (m). HRMS (ESI-TOF) m/z Calcd
for C.sub.8H.sub.9FOPS.sub.3.sup.+ [M+H].sup.+ 266.9532, found
266.9544.
o. 2-(3,5-difluorophenoxy)-1,3,2-dithiaphospholane 2-sulfide
(Compound 2-15)
##STR00395##
[0473] The title compound was prepared using method B and LG-H is
3,5-difluorophenol. .sup.1H NMR (400 MHz, Chloroform-d) .delta.
6.84 (dt, J=7.6, 2.3 Hz, 2H), 6.76-6.66 (m, 1H), 3.84-3.61 (m, 4H),
13C NMR (151 MHz, Chloroform-d) .delta. 163.04 (ddd, J=249.4, 14.9,
2.4 Hz), 152.08 (q, J=13.7 Hz), 106.35 (dd, J=28.6, 5.8 Hz), 101.84
(td, J=25.3, 2.4 Hz), 42.01. 31P NMR (202 MHz, Chloroform-d)
.delta. 119.91 (p, J=16.7, 16.3 Hz). HRMS (ESI-TOF) m/z Calcd for
C.sub.8H.sub.8F.sub.2OPS.sub.3.sup.+ [M+H].sup.+ 284.9437, found
284.9449.
p. 2-(3,5-bis(trifluoromethyl)phenoxy)-1,3,2-dithiaphospholane
2-sulfide (Compound 2-16)
##STR00396##
[0475] The title compound was prepared using method B and LG-H is
3,5-bis(trifluoromethyl)phenol. .sup.1H NMR (500 MHz, Chloroform-d)
.delta. 7.78-7.73 (m, 1H), 7.71 (d, J=1.8 Hz, 2H), 3.88-3.67 (m,
4H), 13C NMR (151 MHz. Chloroform-d) .delta. 151.33 (d, J=12.4 Hz),
133.02 (m), 122.91 (m), 122.83 (d, J=1.8 Hz), 119.64 (q, J=3.7 Hz),
42.12. .sup.31P NMR (202 MHz, Chloroform-d) .delta. 121.42 (t,
J=16.6 Hz). HRMS (ESI-TOF) m/z Calcd for
C.sub.8H.sub.8F.sub.2OPS.sub.3.sup.+ [M+H].sup.+ 284.9437, found
284.9449.
q. 2-(3,4,5-trifluorophenoxy)-1,3,2-dithiaphospholane 2-sulfide
(Compound 2-17)
##STR00397##
[0477] The title compound was prepared using method B and LG-H is
3,4,5-trifluorophenol. .sup.1H NMR (400 MHz, Chloroform-d) .delta.
6.95 (ddd, J=8.0, 5.8, 2.2 Hz, 2H), 3.84-3.64 (m, 4H), 3C NMR (151
MHz, Chloroform-d) .delta. 151.13 (dddd, J=251.3, 11.4, 5.4, 2.6
Hz), 145.45 (m), 138.49 (m), 107.59 (dt, J=19.2, 5.2 Hz), 42.03.
31P NMR (202 MHz, Chloroform-d) .delta. 121.62-120.89 (m). HRMS
(ESI-TOF) m/z Calcd for C.sub.8H.sub.7F.sub.3OPS.sub.3.sup.+
[M+H].sup.+ 302.9343, found 302.9361.
r. 2-(2,4,6-tribromophenoxy)-1,3,2-dithiaphospholane 2-sulfide
(Compound 2-18)
##STR00398##
[0479] The title compound was prepared using method B and LG-H is
2,4,6-tribromophenol. .sup.1H NMR (600 MHz, Chloroform-d) .delta.
7.70 (d, I=0.9 Hz, 2H), 3.84-3.70 (m, 4H). .sup.13C NMR (151 MHz,
Chloroform-d) .delta. 147.43 (d, J=14.6 Hz), 134.93 (d, J=2.7 Hz),
119.84 (d, J=5.5 Hz), 119.16 (d, J=3.9 Hz), 42.16. .sup.31P NMR
(202 MHz, Chloroform-d) .delta. 124.00 (p, J=18.7 Hz). HRMS
(ESI-TOF) m/z Calcd for C.sub.8H.sub.7Br.sub.3OPS.sub.3.sup.+
[M+H].sup.+ 482.6941, found 482.6960.
s. 2-(perchlorophenoxy)-1,3,2-dithiaphospholane 2-sulfide (Compound
2-19)
##STR00399##
[0481] The title compound was prepared using method B and LG-H is
2,3,4,5,6-pentachlorophenol. .sup.1H NMR (600 MHz, Chloroform-d)
.delta. 3.87-3.75 (m, 4H). .sup.13C NMR (151 MHz, Chloroform-d)
.delta. 145.56 (d, J=14.4 Hz), 131.95 (d, J=3.6 Hz), 130.92 (d,
J=3.8 Hz), 128.88 (d, J=5.4 Hz), 42.29. .sup.31P NMR (202 MHz,
Chloroform-d) .delta. 125.13 (m). HRMS (ESI-TOF) m/z Calcd for
C.sub.8H.sub.7Br.sub.3OPS.sub.3.sup.+ [M+H].sup.+ 482.6941, found
482.6960.
t. 2-phenoxy-1,3,2-dithiaphosphinane 2-sulfide (Compound 2-20)
##STR00400##
[0483] The title compound was prepared using method B and LG-H is
phenol and dithiol is propane-1, 3-dithiol. .sup.1H NMR (500 MHz,
Chloroform-d) .delta. 7.41-7.31 (m, 4H), 7.25-7.20 (m, 1H),
3.53-3.41 (m, 2H), 3.17-3.01 (m, 2H), 2.40-2.30 (m, 1H), 2.21-2.09
(m, 1H). .sup.13C NMR (151 MHz, Chloroform-d) .delta. 150.76 (d,
J=12.2 Hz), 129.76 (d, J=2.2 Hz), 125.74 (d, J=2.8 Hz), 121.46 (d,
J=5.8 Hz), 32.63 (d, J=4.5 Hz), 24.43 (d, J=5.0 Hz). .sup.31P NMR
(202 MHz, Chloroform-d) .delta. 82.11 (tt, J=26.5, 8.1 Hz). HRMS
(ESI-TOF) m/z Calcd for C.sub.9H.sub.12OPS.sub.3.sup.+ [M+H].sup.+
262.9782, found 262.9800.
u. 2-(4-bromophenoxy)-1,3,2-dithiaphosphinane 2-sulfide (Compound
2-21)
##STR00401##
[0485] The title compound was prepared using method B and LG-H is
4-bromophenol and dithiol is propane-1, 3-dithiol. .sup.1H NMR (600
MHz, Chloroform-d) .delta. 7.51-7.46 (m, 2H), 7.24-7.20 (m, 2H),
3.48-3.39 (m, 2H), 3.16-3.05 (m, 2H), 2.39-2.31 (m, 1H), 2.20-2.09
(m, 1H). .sup.13C NMR (151 MHz, Chloroform-d) .delta. 149.71 (d,
J=12.2 Hz), 132.78 (d, J=2.1 Hz), 123.28 (d, J=5.8 Hz), 118.89 (d,
J=2.8 Hz), 32.63 (d, J=4.6 Hz), 24.30 (d, J=5.1 Hz). .sup.31P NMR
(202 MHz. Chloroform-d) .delta. 82.85 (ddt, J=26.5, 15.3, 8.0 Hz).
HRMS (ESI-TOF) m/z Calcd for C.sub.9H.sub.11BrOPS.sub.3.sup.+
[M+H].sup.+ 340.8888, found 340.8897.
iii. Method C
[0486] Compound 2-9 was also prepared by Method C. The procedure
below is for the purpose of illustration.
##STR00402##
[0487] Intermediate 2-vii: To a slurry of phosphorus pentasulfide
(20 g, 90 mmol, 1.0 equiv) in dichloromethane (100 mL) was charged
pentafluorophenol (33 g, 180 mmol, 2.0 equiv.) under nitrogen
atmosphere. Triethylamine (27 mL, 189 mmol, 2.1 equiv.) was then
added over a period of 20 min. The mixture was heated at 40.degree.
C., and held at the temperature for 5 h, then cooled to ambient
temperature. A mixture of MTBE and hexanes (1:1, 200 mL) was added.
The resulting mixture was washed with water (200 mL.times.2), and
concentrated to about 60 mL under vacuum. MeOH (100 mL) was added,
and the resulting mixture was concentrated to about 60 mL. MeOH (50
mL) and hexanes (45 mL) were added. Water (30 mL) was then slowly
added over a period of 25 min. The resulting mixture was agitated
at ambient temperature for 1.5 h prior to filtration. The resulting
filter cake was washed with a mixture of MeOH/water (7:3; 30
mL.times.2), then hexanes (25 mL.times.2), providing intermediate
2-vii as white solids (38 g, 76%). M.p.: 103.degree. C. .sup.1H NMR
(400 MHz, CHLOROFORM-d) .delta. 8.79 (s, br, 1H), 3.29 (qd, J=7.3,
5.3 Hz, 6H), 1.42 (t, J=7.3 Hz, 9H). .sup.13C NMR (101 MHz,
CHLOROFORM-d) .delta. 143.3, 140.9, 139.3, 136.8, 46.6, 8.5.
.sup.31P NMR (162 MHz, CHLOROFORM-d) .delta. 114.9 (s, 1P).
.sup.19F NMR (376 MHz, CHLOROFORM-d) .delta. -150.94 (m, 4F),
-161.53 (m, 2F), -164.08 (m, 4F). HRMS (M-H).sup.-: calc. for
C.sub.12F.sub.10O.sub.2PS.sub.2 460.8918, found 460.8919.
[0488] Compound 2-9: To a mixture of 2-vii (10.0 g, 17.8 mmol, 1.0
equiv.) and ethylene sulfide (2.32 mL, 39.1 mmol, 2.2 equiv.) in
dichloromethane (60 mL) was added trifluoroacetic acid (4.08 mL,
53.3 mmol, 3.0 equiv.) at ambient temperature. The reaction mixture
was stirred at the temperature for 16 h, followed by addition of
heptane (120 mL) and water (100 mL). After 20 min agitation, the
mixture was filtered to remove the resulting white solids of
thiirane polymers. The separated organic phase from the filtrate
was treated with 10% aqs. K.sub.2HPO.sub.4 solution (100 mL) and
triethylamine (0.013 mL, 0.5 M %) for 10 min, aqueous phase was
then removed. The organic layer was mixed again with aqs. 10%
K.sub.2HPO.sub.4 solution (150 mL) until completion of the
conversion to the product by HPLC or .sup.31P NMR analysis. The
isolated organic layer was washed with 0.5 M aqs. H.sub.3PO.sub.4
solution (50 mL) and aqs. 10% KH.sub.2PO.sub.4 solution (50 mL),
respectively. The organic stream was then filtered through an
anhydrous MgSO.sub.4 pad, and the resulting filtrate was
concentrated under vacuum to .about.35 mL. Solids were formed
during the concentration. The resulting slurry was cooled to
0.degree. C., agitated for 0.5 h, and filtered. The filter cake was
washed with cold heptane (10 mL.times.2), providing 4.35 g of
Compound 2-9 as white solids in 71% yield.
Example 4
##STR00403##
[0489]
N-(9-((3aR,4R,6R,6aR)-2,2-dimethyl-6-(((2-sulfido-1,3,2-dithiaphosp-
holan-2-yl)oxy)methyl)tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-yl-
)benzamide (Compound 4-46)
[0490]
N-(9-((3aR,4R,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[-
3,4-d][1,3]dioxol-4-yl)-9H-purin-6-yl)benzamide (0.50 mmol) and
2-(4-nitrophenoxy)-1,3,2-dithiaphospholane 2-sulfide (1.00 mmol)
were dissolved in acetonitrile (0.10 M) and cooled to 0.degree. C.
To this was added 4-dimethylaminopyridine (DMAP) (1.00 mmol) and
the reaction was allowed to stir for 1 hour at this temperature.
The acetonitrile was removed in vacuo and the resulting residue was
applied to silica gel. The product was eluted with
dichloromethane/acetone (10:1) affording the title compound (0.29
mmol, 58% yield) as a white solid. The reaction can be represented
by the following scheme:
##STR00404##
Example 5
##STR00405##
[0491]
O-(((3aR,4R,6R,6aR)-6-(6-benzamido-9H-purin-9-yl)-2,2-dimethyltetra-
hydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)O-((2R,3S,5S)-2-(((tert-butyldimet-
hylsilyl)oxy)methyl)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)t-
etrahydrofuran-3-yl) S-hydrogen phosphorodithioate (Compound
5-13)
[0492]
1-((2S,4S,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-hydroxytet-
rahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (1.00 mmol)
and
N-(9-((3aR,4R,6R,6aR)-2,2-dimethyl-6-(((2-sulfido-1,3,2-dithiaphospholan--
2-yl)oxy)methyl)tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-yl)benza-
mide (0.50 mmol) were dissolved in acetonitrile (0.10 M). To this
was added DBU (1.50 mmol) and the reaction was allowed to stir for
1 hour. The acetonitrile was removed in vacuo and the resulting
residue was applied to silica gel. The product was eluted with
dichloromethane/methanol (10:1) affording the title compound (0.34
mmol, 68% yield) as a white solid. The reaction can be represented
by the following scheme:
##STR00406##
Example 6
##STR00407##
[0493]
1-((2S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((4-
,5-dimethyl-2-oxido-1,3,2-oxathiaphospholan-2-yl)oxy)tetrahydrofuran-2-yl)-
-5-methylpyrimidine-2,4(1H,3H)-dione (Compound 3-127)
[0494] To a solution of
1-((2S,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)
methyl)-4-((4,5-dimethyl-2-sulfido-1,3,2-oxathiaphospholan-2-yl)oxy)tetra-
hydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (0.26 mmol) in
acetonitrile (0.10 M) was added selenium dioxide (0.52 mmol). The
reaction was allowed to stir for one hour. The reaction was
filtered through celite and the solvent removed in vacuo affording
the title compound (0.25 mmol, 96% yield) as a white solid. The
reaction can be represented by the following scheme:
##STR00408##
Example 7
##STR00409##
[0495]
N-(tert-butoxycarbonyl)-O-((2S,3aS,5R,7aS)-7a-methyl-5-(prop-1-en-2-
-yl)-2-sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-2-yl)-L-serylglycyl--
L-alanine (Compound 7-8)
[0496] Resin bound peptide was prepared under standard Fmoc-SPPS
conditions. To the resin bound peptide (0.010 mmol) was added
(2S,3aS,5R,7aS)-7a-methyl-2-((perfluorophenyl)thio)-5-(prop-1-en-2-yl)hex-
ahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide (0.10 mmol)
followed by triethylamine (0.10 mmol), DMAP (0.00010 mmol) and
acetonitrile (0.10 M). This was agitated for 2 hours. Following
standard washings and cleavage from resin, the phosphorylated
peptide was isolated upon concentration.
Example 8
##STR00410##
[0497]
O--((S)-(((2R,3S,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl-
)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1
(2H)-yl)tetrahydrofuran-3-yl)oxy)(mercapto)phosphoryl)-N-(tert-butoxycarb-
onyl)-L-serylglycyl-L-alanine (Compound 7-1)
[0498] Resin bound peptide was prepared under standard Fmoc-SPPS
conditions. To the resin bound peptide (0.010 mmol) was added
1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)
methyl)-4-(((2S,3aR,5S,7aR)-7a-methyl-5-(prop-1-en-2-yl)-2-sulfidohexahyd-
robenzo[d][1,3,2]oxathiaphosphol-2-yl)oxy)tetrahydrofuran-2-yl)-5-methylpy-
rimidine-2,4(1H,3H)-dione (0.030 mmol) followed by DBU (0.03 mmol)
and acetonitrile (0.10 M). This was agitated for 3 hours. Following
standard washings and cleavage from resin, the phosphorylated
peptide was isolated upon concentration.
Example 9
Synthesis of Pentamer (Compound 5-15)
##STR00411##
[0499]
O-((2R,3R,4R,5R)-2-((((((2R,3S,4R,5R)-2-((((((2R,3R,4R,5R)-5-(6-ami-
no-7,8-dihydro-9H-purin-9-yl)-2-((((((2R,3
S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofu-
ran-3-yl)oxy)sulfidophosphoryl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl)ox-
y)sulfidophosphoryl)oxy)methyl)-5-(6-amino-9H-purin-9-yl)-4-fluorotetrahyd-
rofuran-3-yl)oxy)sulfidophosphoryl)oxy)methyl)-5-(6-amino-9H-purin-9-yl)-4-
-fluorotetrahydrofuran-3-yl)O-(((2R,3S,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-
-3,4-dihydropyrimidin-1 (2H)-yl)tetrahydrofuran-2-yl)methyl)
phosphorothioate
[0500] Step 1: Synthesis of Dimer 9-iii
##STR00412##
[0501] To a deprotected resin bound dT-Q-CPG (9-i, 5 umol, 98 mg)
in a solid-phase peptide synthesizer (SPPS) vessel was added 20
equiv. of dry reagent P(V) reagent (9-ii). Reagent (9-ii) is made
in a manner similar to the intermediate made in Example 2(a),
substituting a protected deoxyadenine nucleoside for the protected
deoxythymidine. Then 40 equiv. of DBU and MeCN [0.1 M relative to
resin-bound substrate] were drawn into vessel. The vessel was
agitated on a shaker for 6 hours. The resulted resin was
de-protected with DCA/DCM (3:97, vol:vol) to afford a resin bound
dimer 9-iii.
Step 2: Synthesis of Trimer 9-iv
##STR00413##
[0503] The procedure to make a resin bound trimer 9-iv is same as
step 1 above except that the starting material is resin bound dimer
9-iii in this step and the reaction was agitated on a shaker for 12
h.
Step 3: Synthesis of Tetramer 9-v
##STR00414##
[0505] The procedure to make a resin bound tetramer 9-v is same as
step 1 above except that the starting material is resin bound
trimer 9-iv in this step and the reaction was agitated on a shaker
for 5 h.
Step 4: Synthesis of Pentamer (Compound 5-15)
##STR00415##
[0507] The procedure to make resin-bound pentamer (compound 5-15)
is same as step 1 above except that the starting material is resin
bound tetramer 9-v and the reaction was agitated on a shaker for
4.5 h. After resin bound pentamer (compound 5-15) was obtained, it
was cleaved from the resin with NH.sub.4OH (28% aq.) for 5 min. The
crude material was purified on a Waters Autopurification LC with a
Waters BEH C18 column (19.times.150 mm, 5 .mu.m) using a 0.1 M
aqueous ammonium formate:acetonitrile gradient (30 mL/min) at
ambient temperature. HPLC analysis showed an expected mix of dimer,
trimer, tetramer, and pentamer. The HPLC retention time of dimer,
trimer, tetramer, and pentamer is 3.86 min, 4.52 min, 5.54 min,
6.71 min, respectively.
Example 10
Synthesis of Pentamer (Compound 5-16)
##STR00416##
[0508]
O-(2R,3S,5R)-2-((((((2R,3R,5R)-2-((((((2R,3S,5R)-5-(4-amino-2-oxopy-
rimidin-1(2H)-yl)-2-((((((2R,3R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin--
9-yl)-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)sulfidophosphoryl)oxy)meth-
yl)tetrahydrofuran-3-yl)oxy)sulfidophosphoryl)oxy)methyl)-5-(6-amino-9H-pu-
rin-9-yl)tetrahydrofuran-3-yl)oxy)sulfidophosphoryl)oxy)methyl)-5-(5-methy-
l-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl)O-(((2R,3S,-
5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahyd-
rofuran-2-yl)methyl) phosphorothioate (compound 5-16)
##STR00417##
[0510] The procedure to make compound 5-16 is same as the procedure
to make compound 5-15 except that the nucleotide reagents were
changed correspondingly in each step and the reaction to make the
resin bound tetramer was agitated on a shaker for 3 h.
Example 11
[0511] Stereoselective Synthesis of a CDN (ammonium salt of
compound 6-22)
##STR00418##
(2R,3R,3aR,7aR,9R,10aS,14aR)-2-(6-amino-9H-purin-9-yl)-3-fluoro-9-(2-(iso-
butylamino)-6-oxo-1,6-dihydro-9H-purin-9-yl)octahydro-2H,7H-difuro[3,2-d:3-
',2'-j][1,3,7,9]tetraoxa[2,8]diphosphacyclododecine-5,12-bis(thiolate)
5,12-dioxide
[0512] Step 1: Synthesis of CDN Precursor 11-iii
##STR00419##
[0513] 1 eq of 11-i in DMF was mixed with 2 eq. of 11-ii and 4.0
eq. DBU. The mixture was stirred at 25.degree. C. for 10 min. After
the reaction was done, triethylammonium fluoride (10%/v) was added
and the resulted solution was stirred for 12 hours to afford crude
11-iii, which was purified by HPLC. Retention time in HPLC: 3.56
min. MS spectrum confirmed formation of a single isomer (11-iii)
that was also stable to purification.
Step 2: Stereoselective Synthesis of CDN (Ammonium Salt of Compound
6-22)
##STR00420##
[0515] To an MeCN solution [0.1 M] of 11-iii, 3 eq. of 10-3 and
15.0 eq of DBU were added. The resulted solution was stirred at
25.degree. C. for 20 min. After the reaction was done, the mixture
was subject to HPLC purification affording two diastereomers of
CDNs with a ratio of 7:1. The formation of two diastereomers was
confirmed by mass spectrometry.
[0516] In addition to the above reactions, a base and solvent
screen was conducted. It was found that NMI, imidazole, DBU, DMAP
(6.0 eq.) DMAP (1.5 eq.) are all suitable bases for the reactions
and THF, DMF, and MeCN are suitable solvents for the reactions.
Example 12
Synthesis of Organothiophosphates Using P(S).sub.2 Reagent
a.
1-((2R,4S,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-((2-sulfido-1,-
3,2-dithiaphospholan-2-yl)oxy)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4-
(1H,3H)-dione (Compound 4-43)
##STR00421##
[0518] The scheme to prepare compound 4-43:
##STR00422##
[0519] To a solution of 12-i (0.5 mmol) and compound 2-1 (1.0
equiv.) in MeCN [0.1 M], was added 1.5 equiv. of DMAP. The reaction
was stirred at room temperature for 16 h. Isolation by flash column
chromatography (1:5 acetone:DCM) afforded compound 4-43 (0.16 g,
54%). .sup.1H NMR (600 MHz, CDCl.sub.3) .delta. 8.57 (s, 1H),
7.74-7.60 (m, 4H), 7.54-7.34 (m, 7H), 6.46 (s, 1H), 5.55 (s, 1H),
5.30 (s, 1H), 4.31 (s, 1H), 3.97 (s, 2H), 3.66 (s, 4H), 2.61 (s,
1H), 2.29 (s, 1H), 1.61 (d, J=1.2 Hz, 4H), 1.10 (s, 10H). .sup.13C
NMR (151 MHz, CDCl.sub.3) .delta. 163.56, 150.44, 135.76, 135.39,
135.02, 132.85, 131.95, 130.42, 130.27, 128.30, 128.19, 111.80,
85.66, 85.63, 84.55, 79.20, 79.14, 63.90, 41.98, 41.73, 39.44,
39.41, 27.16, 19.54, 12.18. .sup.31P NMR (162 MHz, CDCl.sub.3)
.delta. 122.41 (h, J=15.9 Hz). M.sup.+ 635.189; Rt.: 8.173 min.
b.
1-((2R,4S,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-((2-sulfido-1,-
3,2-dithiaphospholan-2-yl)oxy)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4-
(1H,3H)-dione (Compound 4-1)
##STR00423##
[0521] A vial was flame-dried and allowed to cool under vacuum. The
vial was then placed under inert Ar atmosphere via balloon.
NO.sub.2-dithiaphospholane (compound 2-1, 2.0 equiv, 16.5 mg, 0.056
mmol) and
1-((2R,4S,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-hydroxytetra-
hydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (1.0 equiv,
10.0 mg, 0.028 mmol) were added to the flask. Anhydrous
acetonitrile (0.1M, 0.28 mL) was added, and the mixture was stirred
for 5 min. DMAP (1.5 equiv, 5.15 mg, 0.042 mmol) was added, and the
mixture was stirred at room temperature overnight. The crude was
directly purified by TLC with elution hexanes: DCM (10% DCM in
Hexane) to afford a white solid (8 mg, 56%, Rf: 0.585 (20% DCM in
Hexane)). .sup.1H NMR (600 MHz, CDCl.sub.3) .delta. 8.35 (s, 1H),
7.53 (q, J=1.2 Hz, 1H), 6.42 (dd, J=9.3, 5.2 Hz, 1H), 5.35 (dd,
J=13.9, 5.8 Hz, 1H), 4.41-4.29 (m, 1H), 3.92 (dd, J=7.7, 2.1 Hz,
2H), 3.79-3.62 (m, 4H), 2.53 (dd, J=13.9, 5.3 Hz, 1H), 2.18-2.09
(m, 1H), 1.92 (d, J=1.3 Hz, 3H), 0.94 (s, 9H), 0.15 (d, J=1.0 Hz,
6H). .sup.13C NMR (151 MHz, CDCl.sub.3) .delta. 163.49, 150.33,
135.17, 111.49, 85.91, 85.88, 84.86, 79.80, 79.74, 63.40, 42.05,
41.70, 39.52, 39.48, 26.11, 18.51, 12.65, -5.20. .sup.31P NMR (162
MHz, Acetone) .delta. 122.59.
c. N-(1-((2R,4S,5R)-5-(((tert-butyldimethyl
silyl)oxy)methyl)-4-((2-sulfido-1,3,2-dithiaphospholan-2-yl)oxy)tetrahydr-
ofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (Compound
4-44)
##STR00424##
[0523] A vial was flame-dried and allowed to cool under vacuum. The
vial was then placed under inert Ar atmosphere via balloon.
NO.sub.2-dithiaphospholane (compound 2-1, 2.0 equiv, 13.2 mg,
0.0449 mmol) and
N-(1-((2R,4S,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-hyd-
roxytetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide
(1.0 equiv, 10.0 mg, 0.0225 mmol) were added to the flask.
Anhydrous THF (0.1M, 0.22 mL) was added, and the mixture was
stirred for 5 min. DBU (1.5 equiv, 5.0 .mu.L, 0.0337 mmol) was
added, and the mixture was stirred at rt overnight. The crude was
directly purified by TLC with elution hexanes: DCM (20% DCM in
Hexane) to afford a white solid (6 mg, 44.6%, Rf: 0.451 (20% DCM in
Hexane)). .sup.1H NMR (600 MHz, CDCl.sub.3) .delta. 8.36 (d, J=7.5
Hz, 1H), 7.91 (d, J=7.7 Hz, 2H), 7.62 (t, J=7.5 Hz, 1H), 7.55 (s
broad, 1H), 7.52 (s, J=7.5 Hz, 2H), 6.44 (dd, J=7.9, 5.6 Hz, 1H),
5.34 (ddt, J=13.9, 6.2, 2.0 Hz, 1H), 4.47 (d, J=2.1 Hz, 1H), 3.95
(dd, J=4.6, 2.1 Hz, 2H), 3.78-3.62 (m, 4H), 2.91-2.79 (m, 1H),
2.25-2.14 (m, 1H), 0.92 (s, 9H), 0.14 (d, J=2.1 Hz, 6H). .sup.13C
NMR (151 MHz, CDCl.sub.3) .delta. 162.24, 144.94, 133.45, 129.25,
127.69, 126.32, 115.92, 87.40, 86.80, 86.77, 79.46, 79.40, 63.09,
42.14, 41.59, 41.10, 41.06, 29.85, 26.07, 18.46, -5.27, -5.29.
.sup.31P NMR (.sup.1H decoupled, 162 MHz, CDCl.sub.3) .delta.
122.82-122.28 (m). .sup.31P NMR (.sup.1H decoupled, 162 MHz,
CDCl.sub.3) .delta. 122.61.
d.
N-(9-((2R,4S,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-((2-sulfido-
-1,3,2-dithiaphospholan-2-yl)oxy)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1-
H-purin-2-yl)isobutyramide (Compound 4-45)
##STR00425##
[0525] A vial was flame-dried and allowed to cool under vacuum. The
vial was then placed under inert Ar atmosphere via balloon.
NO.sub.2-dithiaphospholane (compound 2-1, 4.0 equiv, 26 mg, 0.0886
mmol) and Guanine-C(O).sup.iPr (1.0 equiv, 10.0 mg, 0.0222 mmol)
were added to the flask. Anhydrous Acetonitrile (0.1M, 0.22 mL) was
added, and the mixture was stirred for 5 min. DBU (1.5 equiv, 7.0
.mu.L, 0.0332 mmol) was added, and the mixture was stirred at rt
overnight. The crude was directly purified by TLC with elution
hexanes:DCM (20% DCM in Hexane) to afford a transparent oil (5.8
mg, 43%, Rf: 0.35 (20% DCM in Hexane)). .sup.1H NMR (600 MHz,
CDCl.sub.3) .delta. 11.97 (s, 1H), 8.41 (s, 1H), 7.98 (s, 1H), 6.25
(s, 1H), 5.69-5.58 (m, 1H), 4.32 (d, J=3.1 Hz, 1H), 3.85 (d, J=3.0
Hz, 2H), 3.80-3.63 (m, 4H), 2.84 (d, J=6.8 Hz, 1H), 2.74 (d, J=3.8
Hz, 1H), 2.63 (s, 1H), 1.29 (dd, J=6.9, 2.1 Hz, 6H), 0.86 (s, 9H),
0.05 (d, J=21.2 Hz, 6H). .sup.13C NMR (151 MHz, CDCl.sub.3) .delta.
178.18, 155.45, 147.62, 147.45, 137.41, 121.73, 85.84, 85.80,
84.05, 78.76, 78.71, 62.87, 42.02, 41.92, 39.32, 39.29, 36.86,
31.09, 26.05, 19.15, 19.12, 18.52, -5.26, -5.33. .sup.31P NMR
(.sup.1H coupled, 162 MHz, CDCl.sub.3) .delta. 123.16 (dq, J=31.9,
16.0 Hz). .sup.31P NMR (.sup.1H decoupled, 162 MHz, CDCl.sub.3)
.delta. 123.21.
e.
2-(((2R,3S,5R)-5-(6-amino-9H-purin-9-yl)-2-(((tert-butyldimethylsilyl)o-
xy)methyl) tetrahydrofuran-3-yl)oxy)-1,3,2-dithiaphospholane
2-sulfide (Compound 4-19)
##STR00426##
[0527] A vial was flame-dried and allowed to cool under vacuum. The
vial was then placed under inert Ar atmosphere via balloon.
NO.sub.2-dithiaphospholane (compound 2-1, 2.0 equiv, 16 mg, 0.0548
mmol) and Adenine (1.0 equiv, 10.0 mg, 0.0274 mmol) were added to
the flask. Anhydrous dichloromethane (0.1M, 0.27 mL) was added, and
the mixture was stirred for 5 min. DMAP (1.5 equiv, 5.0 mg, 0.0411
mmol) was added, and the mixture was stirred at rt overnight. The
crude was directly purified by TLC with elution hexanes: DCM (40%
DCM in Hexane) to afford a transparent oil (compound 4-19, 7.0 mg,
49%, Rf: 0.207 (40% DCM in Hexane)). .sup.1H NMR (600 MHz,
CDCl.sub.3) .delta. 8.35 (s, 1H), 8.18 (s, 1H), 6.56 (d, J=1.5 Hz,
1H), 5.71 (s, 2H), 5.49 (d, J=14.0 Hz, 1H), 4.43 (d, J=2.1 Hz, 1H),
3.92 (d, J=2.9 Hz, 2H), 3.71 (dd, J=16.0, 1.7 Hz, 4H), 2.85-2.67
(m, 2H), 0.93 (s, 9H), 0.12 (s, 7H). .sup.13C NMR (151 MHz,
CDCl.sub.3) .delta. 155.41, 153.05, 149.86, 138.84, 119.94, 86.26,
86.23, 84.31, 79.87, 79.81, 63.37, 41.92, 41.85, 40.34, 40.31,
26.14, 18.55, -5.20, -5.29. .sup.31P NMR (.sup.1H coupled, 162 MHz,
CDCl.sub.3) .delta. 122.45 (h, J=15.4 Hz). .sup.31P NMR (.sup.1H
decoupled, 162 MHz, CDCl.sub.3) .delta. 122.50.
Example 13(a)
Synthesis of P(V)-DMT-T16 (all R) (Compound 5-17)
##STR00427##
[0529] Synthesis was carried out on a BioAutomation MerMade MM12
oligonucleotide synthesizer on a 2.5 .mu.mole scale using dT-Q-CPG
500 oligonucleotide synthesis resin (Glen Research, 20-2030-XX).
Each synthesis cycle used the following parameters:
TABLE-US-00008 Wait Step Operation Reagents and Solvents time 1
Detritylation 3% Dichloroacetic acid in dichloromethane (1 mL) 90
sec. 2 Detritylation 3% Dichloroacetic acid in dichloromethane (1
mL) 90 sec. 3 ACN Wash Acetonitrile (1.5 mL) 20 sec. 4 ACN Wash
Acetonitrile (1.5 mL) 20 sec. 5 Coupling 0.1M (S)-.PSI.dT in
acetonitrile (0.2 mL, 8 eq) 300 sec. 1.5M
1,8-diazabicyclo[5.4.0]undec-7-ene in acetonitrile (0.1 mL, 60 eq)
6 Coupling 0.1M (S)-.PSI.dT in acetonitrile (0.2 mL, 8 eq) 300 sec.
1.5M 1,8-diazabicyclo[5.4.0]undec-7-ene in acetonitrile (0.1 mL, 60
eq) 7 Coupling 0.1M (S)-.PSI.dT in acetonitrile (0.2 mL, 8 eq) 300
sec. 1.5M 1,8-diazabicyclo[5.4.0]undec-7-ene in acetonitrile (0.1
mL, 60 eq) 8 ACN Wash Acetonitrile (1.5 mL) 20 sec. 9 ACN Wash
Acetonitrile (1.5 mL) 20 sec. 10 Capping 20% acetic anhydride, 30%
2,6-lutidine, 50% acetonitrile (0.4 mL) 60 sec. 20%
N-methylimidazole in acetonitrile (0.4 mL) 11 ACN Wash Acetonitrile
(1.5 mL) 20 sec. 12 ACN Wash Acetonitrile (1.5 mL) 20 sec.
[0530] (S)-.PSI.dT refers to the reagent
1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((2S,3aS-
,6R,7aS)-3a-methyl-6-(prop-1-en-2-yl)-2-sulfidohexahydrobenzo[d][1,3,2]oxa-
thiaphosphol-2-yl)oxy)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)--
dione having the structure:
##STR00428##
[0531] At the completion of the synthesis the column was subjected
to two more acetonitrile washes. The column was removed from the
synthesizer. Air was pulled through the column for 20 minutes. The
resin in the column was treated with saturated aqueous ammonium
hydroxide (3.times.1 mL), eluting into a 20 mL scintillation vial.
The eluent was concentrated in vacuo. The residue was analyzed by
LCMS (Aquity UPLC Oligo BEH, C18, 1.7.mu., 2.1.times.50 mm; Solvent
A: 97.5% water, 2.5% methanol, 0.2M
1,1,1,3,3,3-hexafluoro-2-propanol, 16 mmol triethylamine; Solvent
B: 40% water, 60% methanol, 0.2M 1,1,1,3,3,3-hexafluoro-2-propanol,
16 mmol triethylamine; 10% B isocratic over 1 min, then 10% B to
60% B over 6 min then 60% B to 100% B over 0.5 min and holding at
100% B for 1.5 min, all at 1 mL/min, 65.degree. C.) m/3-=1782 (rf
6.5 min, 34% purity at 262 nm).
Example 13(b)
Synthesis of P(V)-T16 (all R) (Compound 5-18)
##STR00429##
[0533] Synthesis was carried out on a BioAutomation MerMade MM12
oligonucleotide synthesizer on a 2.5 .mu.mole scale using dT-Q-CPG
500 oligonucleotide synthesis resin (Glen Research, 20-2030-XX).
Each synthesis cycle used the following parameters:
TABLE-US-00009 Step Operation Reagents and Solvents Wait time 1
Detritylation 3% Dichloroacetic acid in dichloromethane (1 mL) 90
sec. 2 Detritylation 3% Dichloroacetic acid in dichloromethane (1
mL) 90 sec. 3 ACN Wash Acetonitrile (1.5 mL) 20 sec. 4 ACN Wash
Acetonitrile (1.5 mL) 20 sec. 5 Coupling 0.1M (S)-.PSI.dT in
acetonitrile (0.05 mL, 2 eq) 120 sec. 1M
1,8-diazabicyclo[5.4.0]undec-7-ene in acetonitrile (0.025 mL, 10
eq) 6 Coupling 0.1M (S)-.PSI.dT in acetonitrile (0.05 mL, 2 eq) 120
sec. 1M 1,8-diazabicyclo[5.4.0]undec-7-ene in acetonitrile (0.025
mL, 10 eq) 7 Coupling 0.1M (S)-.PSI.dT in acetonitrile (0.05 mL, 2
eq) 120 sec. 1M 1,8-diazabicyclo[5.4.0]undec-7-ene in acetonitrile
(0.025 mL, 10 eq) 8 Coupling 0.1M (S)-.PSI.dT in acetonitrile (0.05
mL, 2 eq) 120 sec. 1M 1,8-diazabicyclo[5.4.0]undec-7-ene in
acetonitrile (0.025 mL, 10 eq) 9 Coupling 0.1M (S)-.PSI.TdT in
acetonitrile (0.05 mL, 2 eq) 120 sec. 1M
1,8-diazabicyclo[5.4.0]undec-7-ene in acetonitrile (0.025 mL, 10
eq) 10 Coupling 0.1M (S)-.PSI.dT in acetonitrile (0.05 mL, 2 eq)
120 sec. 1M 1,8-diazabicyclo[5.4.0]undec-7-ene in acetonitrile
(0.025 mL, 10 eq) 11 ACN Wash Acetonitrile (1.5 mL) 20 sec. 12 ACN
Wash Acetonitrile (1.5 mL) 20 sec. 13 Capping 20% acetic anhydride,
30% 2,6-lutidine, 50% acetonitrile (0.4 mL) 60 sec. 20%
N-methylimidazole in acetonitrile (0.4 mL) 14 ACN Wash Acetonitrile
(1.5 mL) 20 sec. 15 ACN Wash Acetonitrile (1.5 mL) 20 sec.
[0534] At the completion of the synthesis the column was subjected
to two more detritylation steps followed by two more acetonitrile
washes. The column was removed from the synthesizer. Air was pulled
through the column for 15 minutes. The resin was collected in a 2
dram scintillation vial. Saturated aqueous ammonium hydroxide (2
mL) was added to the vial which was then tightly sealed. The
mixture was held at room temperature for 1 hour, then filtered. The
filter cake was washed twice with 50% ethanol--water and the
filtrate was concentrated in vacuo. The resulting residue was
dissolved in water (1 mL), then purified by preparatory HPLC
(Waters XBridge, C18, 5.mu., 19.times.100 mm; Solvent A: 98% water,
2% methanol, 0.4M 1,1,1,3,3,3-hexafluoro-2-propanol, 16 mmol
triethylamine; Solvent B: 40% water, 60% methanol, 0.4M
1,1,1,3,3,3-hexafluoro-2-propanol, 16 mmol triethylamine; run
gradient 10% B-A to 80% B-A over 25 minutes at 20 mL/min). The
major UV (220 nm) peak fractions were combined. The combined
fractions were concentrated to -10 mL by centrifugal evaporation
and split into two equal portions. To each portion was added 0.1M
sodium hydroxide (1 mL) and 10 mM sodium hydroxide, 2M sodium
chloride (3 mL), and each was partially concentrated by centrifugal
evaporation for 1 hour and then desalted by gel filtration (HiPrep
26/10 Desalting, CV 53 mL.times.2 columns (106 mL); 100% water
isocratic over 1.5 CV). The product containing fractions were
combined, isolated, frozen and lyophilized. The desired product was
isolated as fluffy white solid in 1.3% yield. LCMS (Aquity UPLC
Oligo BEH, C18, 1.7.mu., 2.1.times.50 mm; Solvent A: 97.5% water,
2.5% methanol, 0.2M 1,1,1,3,3,3-hexafluoro-2-propanol, 16 mmol
triethylamine; Solvent B: 40% water, 60% methanol, 0.2M
1,1,1,3,3,3-hexafluoro-2-propanol, 16 mmol triethylamine; 10% B-A
isocratic over 0.5 min then 10% B-A to 35% B-A over 2.25 min then
35% B-A to 100% B over 0.5 min at 1 mL/min, 65.degree. C.)
m/3-=1680.8 (rf 2.74 min, >95% purity).
Example 13(c)
Synthesis of 17-mer TAGTCGACTIGGCCAAT (Compound 5-18)
##STR00430##
[0536] Synthesis was carried out on a BioAutomation MerMade MM12
oligonucleotide synthesizer on a 2.5 .mu.mole scale using dT-Q-CPG
500 oligonucleotide synthesis resin (Glen Research, 20-2030-XX).
The 17-mer TAGTCGACTTGGCCAAT was designed to contain all possible
dinucleotide linkages (i.e. A-C, C-A, A-G, etc.). Each synthesis
cycle used the following parameters:
TABLE-US-00010 Step Operation Reagents and Solvents Wait time 1
Detritylation 3% Dichloroacetic acid in dichloromethane (1 mL) 90
sec. 2 Detritylation 3% Dichloroacetic acid in dichloromethane (1
mL) 90 sec. 3 ACN Wash Acetonitrile (1.5 mL) 20 sec. 4 ACN Wash
Acetonitrile (1.5 mL) 20 sec. 5 Coupling 0.1M (S)-.PSI.-nucleoside
in solvent (0.05 mL, 2 eq) 120 sec. 1M
1,8-diazabicyclo[5.4.0]undec-7-ene in acetonitrile (0.025 mL, 10
eq) 6 Coupling 0.1M (S)-.PSI.-nucleoside in solvent (0.05 mL, 2 eq)
120 sec. 1M 1,8-diazabicyclo[5.4.0]undec-7-ene in acetonitrile
(0.025 mL, 10 eq) 7 Coupling 0.1M (S)-.PSI.-nucleoside in solvent
(0.05 mL, 2 eq) 120 sec. 1M 1,8-diazabicyclo[5.4.0]undec-7-ene in
acetonitrile (0.025 mL, 10 eq) 8 Coupling 0.1M (S)-.PSI.-nucleoside
in solvent (0.05 mL, 2 eq) 120 sec. 1M
1,8-diazabicyclo[5.4.0]undec-7-ene in acetonitrile (0.025 mL, 10
eq) 9 Coupling 0.1M (S)-.PSI.-nucleoside in solvent (0.05 mL, 2 eq)
120 sec. 1M 1,8-diazabicyclo[5.4.0]undec-7-ene in acetonitrile
(0.025 mL, 10 eq) 10 Coupling 0.1M (S)-.PSI.-nucleoside in solvent
(0.05 mL, 2 eq) 120 sec. 1M 1,8-diazabicyclo[5.4.0]undec-7-ene in
acetonitrile (0.025 mL, 10 eq) 11 ACN Wash Acetonitrile (1.5 mL) 20
sec. 12 ACN Wash Acetonitrile (1.5 mL) 20 sec. 13 Capping 20%
acetic anhydride, 30% 2,6-lutidine, 50% acetonitrile (0.4 mL) 60
sec. 20% N-methylimidazole in acetonitrile (0.4 mL) 14 ACN Wash
Acetonitrile (1.5 mL) 20 sec. 15 ACN Wash Acetonitrile (1.5 mL) 20
sec.
[0537] Wherein "(S)-.PSI.-nucleoside in solvent" refers to:
[0538] for deoxyadenosine: compound 3-128 (listed in Example 16) in
acetonitrile;
[0539] for deoxy(5-methyl)cytosine: compound 3-129 (listed in
Example 16) in acetonitrile;
[0540] for deoxyguanosine: compound 3-130 (listed in Example 16) in
isobutyronitrile;
[0541] for deoxythymidine: compound 3-131 (listed in Example 16) in
acetonitrile.
[0542] Following completion of the synthesis, the column containing
the support was removed from the synthesizer, and dried by pulling
a vacuum through it for a few minutes. The column was treated with
concentrated ammonium hydroxide (3.times.1 mL) to cleave the linker
and the eluent was collected in a red-capped pressure-rated vial.
The vial was heated to 55 C overnight to cleave the base protecting
groups. The mixture was filtered and the filtrate evaporated on a
Biotage V10 evaporator. The sample was analyzed by LC/MS (A
solvent=97.5% Water/2.5% MeOH w/0.2M HFIP/0.016M TEA, B Solvent=40%
Water/60% MeOH w/0.2M HFIP/0.016M TEA) on an Acquity UPLC Oligo BEH
C18 2.1.times.50 MM 1.7 um column, eluting with a gradient 10-100%
over 3.25 min. Ions for M-3/3 (1932.45), M-4/4 (1449.2) and
M-5/5(1159.05) were observed, indicating the presence of the 17-mer
as a major component.
Example 14 (a)
Preparation of Styrene Oxide (S)--P(V) Reagent (Compound 1-13)
##STR00431##
[0544] To a 2.5-L Chemglass reactor was added phosphate TEA salt
(100 g, 1.0 eq), hexanes (1 L) and isopropyl acetate (500 mL).
(R)-styrene oxide (28.3 g, 1.4 eq) was then added, followed by
dichloroacetic acid (86.6 g, 4 eq). The resulting mixture was
stirred at ambient temperature until the reaction was complete. The
mixture was then washed with hexanes (200 mL) and water (400 mL).
The separated organic phase was then washed with 10% aqueous
KH.sub.2PO.sub.4 solution (400 mL), and passed through a MgSO.sub.4
pad. The resulting filtrate was then solvent-swapped into MeOH.
After the volume of batch was adjusted to 0.35 L, the batch was
cooled to 0.degree. C. and stirred until a seed bed was formed.
Water (20 mL) was added into the batch, and the resulting slurry
was stirred for 1 h at 0.degree. C. before filtration. The filter
cake was washed with cold isopropyl alcohol (0.degree. C., 60 mL),
and dried in vacuum. The product was isolated in 45-53% yield with
98 AP and 99 ee %.
[0545] Recrystallization: The dry cake (20 g) was dissolved in
dichloromethane (80 mL) at ambient temperature. The solution was
solvent-swapped into heptane, and the final batch volume was
adjusted to about 60 mL. The resulting mixture was cooled to
0.degree. C., stirred for 1 h, and filtered. The filtrate was
recycled to the reactor to complete the transfer of the slurry. The
cake was then washed with cold heptane (0.degree. C. 20 mL), and
dried at 50.degree. C. under vacuum. The recovery yield was
typically 90%. UHPLC retention time is 1.81 min. Chiral HPLC
retention time is 14.60 min.
Example 14 (b)
Preparation of Styrene Oxide (R)--P(V) Reagent (Compound 1-14)
##STR00432##
[0547] The procedure to make styrene oxide (R)--P(V) reagent
(compound 1-14) is same as the procedure to make compound 1-13
except that styrene oxide in the reaction is a (S)-isomer. Chiral
HPLC retention time for compound 1-14 is 12.74 min.
[0548] Diastereomers of compounds 1-13 and 1-14 were a minor
product of each reaction. Chiral HPLC retention time of the
diastereomer from (S)-styrene oxide is 12.09 min. Chiral HPLC
retention time of the diastereomer from (R)-styrene oxide is 12.42
min.
Example 15
Preparation of Phosphodiester Products
##STR00433##
[0550] In a similar fashion to the phosphorous thiosulfates,
phosphodiester products can be prepared. In this case, reagents
such as compounds 1-10 or 1-11, shown above, were used. A
phosphorous (V) reagent containing a leaving group is loaded onto
the first coupling partner (for example, a nucleoside such as
15-ii) under basic conditions. This affords a loaded compound as an
inconsequential mixture of diastereomers at phosphorous (such as
15-iii). This loaded compound is then coupled with the next
coupling partner under basic conditions (for example, a nucleoside
such as 15-iv) to afford the coupled phosphodiester product
(compound 5-14). Detailed procedures for the preparation of
dinucleotides are listed below.
a. Reagent Preparation (Compound 1-10)
[0551] To a solution of phosphoryl chloride (0.173 g, 1.00 equiv.,
1.15 mmol) in toluene (3.5 mL) was added 3-mercapto-1-propanol
(0.100 mL, 1.15 mmol), followed by triethylamine (0.32 mL, 2.00
equiv., 2.30 mmol) at ambient temperature. The reaction mixture was
stirred for 1 h, and TEA (0.16 mL, 1.0 equiv) was added, followed
by 4-nitrophenol (0.160 g, 1.00 equiv., 1.15 mmol,). After mixing
for 16 h, the reaction mixture was filtered. The reactor was rinsed
with toluene (3 mL), and the rinse was applied to the cake wash.
The combined filtrates were concentrated, and the resulting residue
was purified by silica gel chromatography, providing compound 1-10
as white solids (81 mg, 25%). .sup.1H NMR (400 MHz, CHLOROFORM-d)
.delta. 8.30 (d, J=8.8 Hz, 2H), 7.48 (dd, J=8.8, 1.1 Hz, 2H),
4.77-4.67 (m, 2H), 3.25-3.14 (m, 1H), 3.13-2.97 (m, 1H), 2.45-2.24
(m, 1H), 2.19-2.07 (m, 1H). LCMS m/z (M+H).sup.4 276.
b. Reagent Preparation (Compound 1-11)
[0552] To a solution of 3-mercapto-1-propanol (0.500 mL, 5.75 mmol)
in toluene (15 mL) was added phosphoryl chloride (0.865 g, 1.00
equiv., 5.75 mmol), followed by TEA (1.78 mL, 2.20 equiv., 12.7
mmol) at 0.degree. C. The reaction mixture was stirred at 0.degree.
C. for 1 h, then warmed to ambient temperature and mixed for 1 h.
TEA (0.89 mL, 1.1 equiv.) was added, followed by
pentafluorothiophenol (1.15 g, 1.0 equiv., 5.75 mmol). The
resulting mixture was stirred for 6 h, and filtered. The filtrate
was concentrated, and the resulting residue was purified by silica
gel chromatography, providing compound 1-11 as white solids (0.23
g, 13%). .sup.1H NMR (500 MHz, CHLOROFORM-d) .delta. 4.83-4.60 (m,
2H), 3.48-3.32 (m, 1H), 3.25-3.07 (m, 1H), 2.37-2.22 (m, 1H),
2.19-2.08 (m, 1H). LCMS m/z (M+H).sup.+ 337.
c. Loading with Compound 1-10
[0553] To a solution of compound 1-10(80 mg, 1.5 equiv., 0.29 mmol)
and 15-ii (85 mg, 0.18 mmol) in THF (1.7 mL) was added
1,8-diazabicyclo[5.4.0]undec-7-ene (0.08 mL, 3.1 equiv., 0.5 mmol)
at ambient temperature. After stirring for 16 h, the reaction
mixture was concentrated. The resulting residue was purified by
silica gel chromatography, providing 15-iii as white solids (45 mg,
41%). .sup.1H NMR (500 MHz, CHLOROFORM-d) .delta. 8.67-8.42 (m,
1H), 7.67-7.51 (m, 4H), 7.47-7.26 (m, 7H), 6.48-6.28 (m, 1H),
5.35-5.12 (m, 1H), 4.59-4.35 (m, 2H), 4.33-4.13 (min, 1H),
4.00-3.77 (m, 2H), 3.07-2.76 (m, 2H), 2.67-2.52 (m, 1H), 2.33-2.05
(m, 2H), 2.02-1.84 (m, 1H), 1.52 (d, J=16.8 Hz, 3H), 1.03 (s, 9H).
LCMS m/z (M+H).sup.+ 617.
d. Loading with Compound 1-11
[0554] To a solution of compound 1-11 (50 mg, 2 equiv., 0.15 mmol)
and 15-ii (35 mg, 0.073 mmol) in acetonitrile (0.7 mL) was added
1,8-diazabicyclo[5.4.0]undec-7-ene (0.022 mL, 2.0 equiv., 0.15
mmol) at 0.degree. C. After 20 min, the reaction mixture was
concentrated. The resulting residue was purified by silica gel
chromatography, providing 15-iii as white solids (30 mg, 66%).
e. Coupling
[0555] To a solution of 15-iii (20 mg, 0.032 mmol) and 15-iv (31
mg, 2.0 equiv., 0.065 mmol) in THF (0.6 mL) was added a solution of
potassium t-butoxide in THF (1.0 M, 0.11 mL, 3.5 equiv.) at ambient
temperature. After 20 min, acetic acid (10 .mu.L, 5 equiv.) was
added, and the reaction mixture was concentrated. The resulting
residue was purified by silica gel chromatography, providing
compound 5-14 as white solids (17 mg, 53%). LCMS m/z (M+H).sup.1
1023.
Example 16
Synthesis of Loaded Nucleosides
1. Synthesis of 5'-O-Protected Nucleosides (General Procedure
1)
##STR00434##
[0557] All 5'-OTBDPS nucleosides were prepared from the
commercially available compounds according to the following
procedures.
1.1 Intermediate 16-i
##STR00435##
[0559] 2'-Deoxyadenosine (15.0 g, 58.5 mmol, 1.0 equiv.) and
imidazole (9.96 g, 146 mmol, 2.5 equiv.) were dissolved in dry
pyridine (100 mL) and concentrated in vacuo. The resulting mixture
was dissolved in anhydrous DMF (75 mL). TBDPSCl (16.3 mL, 61.4
mmol, 1.1 equiv.) was added, and the mixture was stirred for 2 h.
The solution was concentrated in vacuo then dissolved in EtOAc (500
mL), and washed with water (2.times.250 mL), then brine (250 mL).
The organic layer was dried over MgSO.sub.4, filtered, and
concentrated in vacuo to afford a thick oil that was purified by
flash column chromatography over silica gel (0% to 5% MeOH in
EtOAc) to afford the product as a white solid 16-1(18.0 g, 63%)
spectral characteristics consistent with the literature
(Krishnakumar, K., et al., Synlett 7, 1055-1058 (2010)).
1.2 Intermediate 16-ii
##STR00436##
[0561] Intermediate 16-ii prepared from 2'-deoxycytidine (15.0 g)
under the same protocol as intermediate 16-iv. Following completion
of the reaction as assessed by TLC, the mixture was concentrated in
vacuo. The crude syrup was dissolved in EtOAc (500 mL), washed with
1 N HCl (150 mL, final pH=6), washed with sodium phosphate (100 mL,
1.0 M, pH=7) buffer, then brine (200 mL). The organics were dried
over MgSO.sub.4, filtered, and concentrated to afford a thick oil.
The oil was purified by flash column chromatography (0% to 10% MeOH
in DCM). The isolated foam was broken down into MTBE/hexane (1:1
v/v, 200 mL) then filtered and the solid was dried at 50.degree. C.
at 20 torr until constant weight was achieved. 16-ii (20.0 g, 66%)
was isolated as a white solid.
[0562] Physical State: White solid;
[0563] .sup.1H NMR (600 MHz, DMSO-d): .delta. 7.70 (d, J=7.4 Hz,
1H), 7.64 (dq, J=6.6, 1.2 Hz, 4H), 7.52-7.41 (m, 6H), 7.17 (s, 1H),
7.13 (s, 1H), 6.18 (t, J=6.5 Hz, 1H), 5.56 (d, J=7.4 Hz, 1H), 5.29
(d, J=4.5 Hz, 1H), 4.29 (dq, J=6.0, 3.9 Hz, 1H), 3.89-3.82 (m, 1H),
3.85 (s, 1H), 3.78-3.71 (m, 1H), 2.19 (ddd, J=13.2, 6.3, 4.1 Hz,
1H), 1.99 (dt, J=13.2, 6.5 Hz, 1H), 1.01 (s, 9H); .sup.13C NMR (151
MHz, DMSO-d.sub.6): .delta. 165.26, 154.67, 140.49, 135.15, 134.99,
132.69, 132.38, 130.02, 129.98, 127.98, 93.83, 86.35, 84.68, 69.77,
63.82, 40.51, 26.84, 26.67, 18.81; HRMS (ESI-TOF): calcd. for
C.sub.25H.sub.32N.sub.3O.sub.8Si [M+H].sup.+ 466.2156; found
466.2157.
1.3 Intermediate 16-iii
##STR00437##
[0564] 2'-Deoxyguanosine was first converted to the dimethylformyl
protected compound to increase solubility in pyridine according to
the procedure described in Huang, H., et al., J. Am. Chem. Soc.
133, 20357-20368 (2011). To a solution of 2'-deoxyguanosine (30.0
g, 112 mmol, 1.0 equiv.) in methanol (300 mL) was added
N,N-dimethylformamide dimethyl acetal (60 mL). After stirring for
72 h, the reaction was filtered and washed with MeOH (200 mL). To a
solution of the DMF protected intermediate (34.4 g, 107 mmol, 1.0
equiv.) and DMAP (2.63 g, 21.4 mmol, 0.2 equiv.) in dry pyridine
(344 mL) was added TBDPSCl (32.6 mL, 123 mmol, 1.2 equiv.). After
stirring for 24 h the reaction was concentrated in vacuo. The
residue obtained was partitioned between EtOAc (500 mL) and HCl (1
N, 500 mL, final pH=6). The organic layer was washed with brine
(500 mL), dried over Na2SO4, filtered, then concentrated. The crude
oil was purified by flash column chromatography (0% to 10% MeOH in
DCM). The resulting solids obtained were stirred with MTBE for 1 h,
filtered then dried to constant weight. The stir/filter protocol
was repeated twice. 16-iii (40.0 g g, 67%) was isolated spectral
characteristics consistent with the literature Hutter, D., et al.,
Nucleosides Nucleotides Nucleic Acids 29, 879-895 (2010).
1.4 Intermediate 16-iv
##STR00438##
[0566] To a solution of 2'-deoxythymidine (15.0 g, 61.3 mmol, 1.0
equiv.) and DMAP (1.51 g, 12.26 mmol, 0.2 equiv.) in dry pyridine
(75 mL) was added TBDPSCl (18.7 mL, 70.5 mmol, 1.2 equiv.). The
mixture was stirred for 16 h. then diluted with EtOAc (200 mL) and
washed with water (200 mL), ammonium chloride (saturated aqueous,
100 mL), then brine (100 mL). The organic layer was dried over
MgSO.sub.4, filtered, then concentrated in vacuo to afford a
residue that was purified by flash column chromatography over
silica gel (25% EtOAc in hexanes to 100% EtOAc). Residual pyridine
was removed by stirring in MTBE/hexanes (1:1, v/v, 150 mL). The
resulting solid was isolated by filtration and washed with MTBE
(100 mL). The white solid was dried at 50.degree. C. and 20 torr
until constant weight was achieved, 16-iv (22.59 g, 77%) was
isolated with spectral characteristics consistent with the
literature (Nagaya, Y., et al., Nucleosides Nucleotides Nucleic
Acids 35, 64-75 (2016)).
1.5 Intermediate 16-v
##STR00439##
[0568] 2'-Deoxy-2'-fluoroadenosine (2.10 g, 7.81 mmol, 1.0 equiv.)
was dried by co-evaporation with anhydrous pyridine (20 mL.times.3)
in vacuo then dissolved in anhydrous DMF (40 mL, 0.2 M) in a
flame-dried round bottom flask. Imidazole (1.17 g, 17.2 mmol, 2.2
equiv.) was added, followed by TBSCl (1.29 g, 8.60 mmol, 1.1
equiv.). The reaction was stirred overnight at ambient temperature.
The reaction was quenched on addition of water (40 mL). After
stirring for 20 minutes, the solids were collected by filtration
and washed with water (40 mL). After drying in vacuo overnight,
16-v (2.54 g, 85%) was isolated as a white solid with spectral
characteristics consistent with the literature (Wnuk, S., et al.,
J. Org. Chem. 67, 8794-8797 (2002)).
2. Synthesis of Loaded Nucleosides (General Procedure 2)
##STR00440##
[0570] The loaded nucleosides were prepared as follows. Nucleoside
(1.0 equiv.) and (+) or (-)-.PSI. (1.3 equiv.) were dissolved in
anhydrous acetonitrile (0.1 M) in a flame-dried round-bottom flask.
DBU (1.3 equiv.) was added dropwise to the reaction mixture while
stirring. Reaction progress was monitored by .sup.31P NMR. After 30
minutes, the crude reaction mixture was filtered through a short
pad of silica gel (approximately 1 inch); then the silica gel was
washed with EtOAc (4.times.5 mL). The filtrate was washed with
saturated aqueous NaHCO.sub.3 (30 mL), water (30 mL), saturated
aqueous KH.sub.2PO.sub.4 (30 mL), dried over MgSO.sub.4 and the
solvent was removed in vacuo. The crude product was purified by
silica gel column chromatography.
[0571] (+)-.PSI. refers to
(2R,3aR,6S,7aR)-3a-methyl-2-((perfluorophenyl)thio)-6-(prop-1-en-2-yl)hex-
ahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide, shown below.
##STR00441##
[0572] (-)-.PSI. refers to
(2S,3aS,6R,7aS)-3a-methyl-2-((perfluorophenyl)thio)-6-(prop-1-en-2-yl)hex-
ahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide, shown below.
##STR00442##
2.1. Compound 3-107
##STR00443##
[0574] To a 250 mL flask were added nucleoside
(2R,3S,5R)-5-(6-amino-9H-purin-9-yl)-2-(((tert-butyldiphenylsilyl)oxy)met-
hyl)tetrahydrofuran-3-ol (intermediate 16-1) 4.14 g, 8.46 mmol, 1
equiv.) and the (+)-.psi. reagent (4.93 g, 11.0 mmol, 1.3 equiv.)
in THF (42 mL). Then, DBU (1.60 mL, 10.6 mmol, 1.3 equiv.) was
added dropwise at ambient temperature. After 20 minutes, the
reaction was quenched with AcOH (1.5 mL, 26 mmol, 3.1 equiv.) and
stirred vigorously under air for 5 h. The crude mixture was
filtered and concentrated to a paste that was purified by flash
column chromatography (0% to 100% EtOAc in DCM) to afford the
desired compound 3-107 as a white solid (6.01 g, 97% yield).
[0575] Physical State: White solid;
[0576] .sup.1H NMR (400 MHz, Chloroform-d): .delta. 8.23 (s, 1H),
8.07 (s, 1H), 7.76-7.54 (m, 4H), 7.48-7.31 (m, 6H), 6.87 (hr s,
2H), 6.50 (t, J=7.1 Hz, 1H), 5.70-5.59 (m, 1H), 5.04 (s, 1H), 4.89
(s, 1H), 4.48 (dt, J=12.6, 3.2 Hz, 1H), 4.41-4.33 (m, 1H),
3.98-3.89 (m, 2H), 2.88-2.73 (m, 2H), 2.59 (br s, 1H), 2.27 (br d,
J=13.1 Hz, 1H), 2.17-2.07 (m, 3H), 1.97-1.80 (m, 2H), 1.78 (s, 3H),
1.71 (s, 3H), 1.07 (s, 9H);
[0577] .sup.13C NMR (101 MHz, Chloroform-d): .delta. 155.4, 152.0,
149.3, 144.8, 138.5, 135.6, 135.4, 132.6, 132.2, 128.98, 128.97,
127.9, 119.1, 112.0, 86.4 (d, J=3.4 Hz), 86.0, 84.3, 79.6 (J=8.0
Hz), 65.7, 63.6, 39.6 (d, J=7.3 Hz), 38.8, 33.8, 33.7, 27.8, 27.6,
26.9, 23.4, 22.7, 21.7, 19.2;
[0578] .sup.31P NMR (162 MHz, Chloroform-d): .delta. 101.0;
[0579] HRMS (ESI-TOF, m/z): HRMS (ESI) Calcd for
[C.sub.36H.sub.46N.sub.5O.sub.4PS.sub.2Si+H].sup.+736.2571; Found
736.2584 (1.7 ppm error).
[0580] R.sub.f=0.45 (25% EtOAc in DCM); UV, KMnO.sub.4.
2.2. Compound 3-108
##STR00444##
[0582] To a 250 mL flask were added nucleoside
(2R,3S,5R)-5-(6-amino-9H-purin-9-yl)-2-(((tert-butyldiphenylsilyl)oxy)met-
hyl)tetrahydrofuran-3-ol (intermediate 16-i) (3.89 g, 7.95 mmol, 1
equiv.) and the (-)-.psi. reagent (4.61 g, 10.3 mmol, 1.3 equiv.)
in THF (40 mL). DBU (1.50 mL, 9.9 mmol, 1.3 equiv.) was then added
dropwise at ambient temperature. After 20 minutes, the reaction was
quenched with AcOH (1.4 mL, 24 mmol, 3.0 equiv.) and stirred
vigorously under air for 5 h. The crude was filtered and
concentrated to a paste that was purified by flash column
chromatography (0% to 100% EtOAc in DCM) to afford the desired
compound 3-108 as a white solid (5.59 g, 96% yield).
[0583] Physical State: White solid;
[0584] M.P. 181.6.degree. C.
[0585] .sup.1H NMR (400 MHz, Chloroform-d): .delta. .delta. 8.31
(s, 1H), 8.05 (s, 1H), 7.72-7.59 (m, 4H), 7.45-7.33 (m, 6H), 6.50
(dd, J=8.6, 5.6 Hz, 1H), 6.07 (s, 2H), 5.64 (br dd, J=11.4, 5.3 Hz,
1H), 5.06 (s, 1H), 4.92 (s, 1H), 4.49 (dt, J=12.5, 3.1 Hz, 1H),
4.36-4.29 (m, 1H), 3.91 (d, J=3.3 Hz, 2H), 2.87-2.70 (m, 2H), 2.60
(br s, 1H), 2.33 (br d, J=13.1 Hz, 1H), 2.19-2.08 (m, 1H),
2.02-1.83 (m, 3H), 1.83-1.69 (m, 7H), 1.07 (s, 9H);
[0586] .sup.13C NMR (101 MHz, Chloroform-d): .delta. 155.5, 152.9,
149.7, 144.7, 138.3, 135.6, 135.4, 132.6, 132.3, 129.95, 129.92,
127.9, 119.8, 112.1, 85.96 (d, J=6.6 Hz), 85.95, 83.8, 79.1 (d,
J=7.3 Hz), 65.8, 63.6, 39.7 (d, J=4.4 Hz), 38.8, 33.7, 33.6, 27.8,
27.6, 26.9, 23.3, 22.7, 21.7, 19.2;
[0587] .sup.31P NMR (162 MHz, Chloroform-d): .delta. 101.1 (s,
1P);
[0588] HRMS (ESI-TOF, m/z): HRMS (ESI) Calcd for
[C.sub.36H.sub.46N.sub.5O.sub.4PS.sub.2Si+H].sup.+ 736.2571; Found
736.2581 (1.4 ppm error).
[0589] R.sub.f=0.35 (25% EtOAc in DCM); UV, KMnO.sub.4.
2.3. Compound 3-109
##STR00445##
[0591] To a solution of nucleoside
4-amino-1-((2R,4S,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-hydroxyt-
etrahydrofuan-2-yl)pyrimidin-2(1H)-one (intermediate 16-ii) (3.20
g, 6.89 mmol, 1.0 equiv) and (-)-.psi. reagent (4.20 g, 9.41 mmol,
1.5 equiv) and in THF (46 mL) was added DBU (1.35 mL, 8.98 mmol,
1.35 equiv) at 0.degree. C. After 1 h, the mixture was diluted with
EtOAc, DCM, and heptane (60 mL each). The mixture was then washed
with K.sub.2HPO.sub.4 (10% aq., 65 mL then 35 mL). The organic
layer was dried over MgSO.sub.4, and concentrated. The resulting
residue was purified by flash chromatography (0 to 10% MeOH in DCM)
to afford the desired compound 3-109 as a white solid (4.3 g, 87%
yield).
[0592] Physical State: White solid;
[0593] .sup.1H NMR (400 MHz, Chloroform-d): .delta. 7.94-7.85 (m,
1H), 7.71-7.62 (m, 4H), 7.54-7.34 (m, 7H), 6.49-6.42 (m, 1H),
5.58-5.46 (m, 2H), 5.05 (s, 1H), 4.88 (s, 1H), 4.45 (dt, J=12.4,
3.2 Hz, 1H), 4.27 (br d, J=2.0 Hz, 1H), 4.04-3.92 (m, 2H), 2.74
(ddd, J=14.1, 5.6, 1.8 Hz, 1H), 2.58 (br s, 1H), 2.31-2.09 (m, 3H),
2.04-1.74 (m, 8H), 1.70 (s, 3H), 1.08 (s, 9H);
[0594] .sup.13C NMR (101 MHz, Chloroform-d): .delta. 144.6, 141.4,
135.7, 135.4, 132.8, 131.9, 130.2, 130.0, 128.1, 128.0, 112.2,
94.3, 86.1, 79.1, 65.6, 63.6, 40.3, 38.8, 33.8, 27.8, 27.0, 23.4,
22.6, 21.7, 19.3;
[0595] .sup.31P NMR (162 MHz, Chloroform-d): .delta. 101.4;
[0596] HRMS (ESI-TOF, m/z): HRMS (ESI) Calcd for
[C.sub.35H.sub.46N.sub.3O.sub.5PS.sub.2Si+H].sup.+712.2459; Found
712.2470 (1.6 ppm error).
[0597] R.sub.f=0.52 (10% MeOH in DCM); UV, KMnO.sub.4.
2.4. Compound 3-110
##STR00446##
[0599] To a solution of nucleoside
4-amino-1-((2R,4S,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-hydroxyt-
etrahydrofuran-2-yl)pyrimidin-2(1H)-one (intermediate 16-ii) (3.5
g, 7.5 mmol, 1.0 equiv) and (+)-.psi. reagent (4.9 g, 11 mmol, 1.5
equiv) in THF (60 mL, 20 v) was added DBU (1.52 mL, 10.1 mmol, 1.35
equiv) at 0.degree. C. After 1 h, the mixture was diluted with
EtOAc, DCM, and heptane (each 60 mL). The mixture was then washed
with K.sub.2HPO.sub.4 (10% aq., 65 mL then 35 mL. The organic layer
was dried over MgSO.sub.4, and concentrated. The resulting residue
was purified by flash chromatography (0 to 10% MeOH in DCM) to
afford the desired compound 3-110 as a white solid (4.2 g, 82%
yield).
[0600] Physical State: White solid;
[0601] .sup.1H NMR (400 MHz, Chloroform-d): .delta. 7.93 (d, J=7.3
Hz, 1H), 7.67 (ddd, J=7.8, 3.9, 1.5 Hz, 4H), 7.53-7.34 (m, 7H),
6.45 (dd, J=7.3, 5.8 Hz, 1H), 5.60-5.33 (m, 3H), 5.08 (s, 1H), 4.92
(s, 1H), 4.47 (dt, J=12.6, 3.3 Hz, 1H), 4.23 (br d, J=2.5 Hz, 1H),
4.02-3.90 (m, 2H), 2.73 (ddd, J=14.0, 5.7, 2.5 Hz, 1H), 2.63-2.57
(m, 1H), 2.35 (br d, J=11.6 Hz, 1H), 2.28-2.10 (m, 2H), 2.10-1.95
(m, 1H), 1.94-1.86 (m, 2H), 1.85-1.77 (m, 4H), 1.71 (s, 3H), 1.08
(s, 9H);
[0602] .sup.13C NMR (101 MHz, Chloroform-d): .delta. 165.1, 155.4,
144.6, 141.1, 135.7, 135.4, 132.8, 132.0, 130.1, 130.0, 128.0 (d,
J=10.9 Hz, 1C), 112.2, 94.2, 85.7, 85.7 (d, J=6.4 Hz, 1C), 78.3 (d,
J=7.3 Hz, 1C), 65.8, 63.4, 40.4 (d, J=4.5 Hz, 1C), 38.8, 33.7 (d,
J=9.1 Hz, 1C), 27.7 (d, J=15.4 Hz, 1C), 27.0, 23.4, 22.7, 21.8,
19.2;
[0603] .sup.31P NMR (162 MHz, Chloroform-d): .delta. 101.5;
[0604] HRMS (ESI-TOF, m/z): HRMS (ESI) Calcd for
[C.sub.35H.sub.46N.sub.3O.sub.5PS.sub.2Si+H].sup.+ 712.2459; Found
712.2473 (2.0 ppm error).
[0605] R.sub.f=0.55 (10% MeOH in DCM); UV, KMnO.sub.4.
2.5. Compound 3-iii
##STR00447##
[0606] A suspension of nucleoside
1-((2R,4S,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-hydroxytetrahydr-
ofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (intermediate
16-iv) (4.00 g, 8.32 mmol, 1.00 equiv.) and the (+)-.psi. reagent
(4.83 g, 10.8 mmol, 1.3 equiv.) in MeCN (83 mL) was cooled to an
internal temperature of 0.degree. C. DBU (1.63 mL, 10.8 mmol, 1.3
equiv.) was added in one portion and stirred at 0.degree. C. for 30
min. The resulting mixture was passed through a plug of silica gel
(ca. 1'') and washed with ethyl acetate (82 mL). The organic layer
was washed with water (42 mL) then NaH.sub.2HPO.sub.4 (10 wt % aq.,
42 mL). The organic layer was dried over MgSO.sub.4, filtered and
concentrated in vacuo to afford a yellow gel that was purified by
flash column chromatography (10% to 90% EtOAc in hexanes) to afford
a foam which was broken down into a powder by stirring in hexane
(3.times.50 mL). This process was repeated three times total. The
compound 3-111 was isolated as a white powder which was dried at
50.degree. C. and 20 Torr until constant weight was reached (5.76
g, 95 wt %, remainder hexanes and EtOAc, 90% corrected yield).
[0607] Physical State: White solid;
[0608] .sup.1H NMR (400 MHz, Chloroform-d): .delta. 8.19 (br s,
1H), 7.64-7.75 (m, 4H), 7.38-7.55 (m, 7H), 6.47 (dd, J=9.5, 5.2 Hz,
1H), 5.60 (dd, J=11.1, 6.1 Hz, 1H), 5.07 (s, 1H), 4.90 (s, 1H),
4.48 (dt, J=12.4, 3.3 Hz, 1H), 4.27 (s, 1H), 3.92-4.09 (m, 2H),
2.51-2.65 (m, 2H), 2.16 (td, J=13.3, 3.9 Hz, 1H), 1.75-2.05 (m,
4H), 1.79 (s, 3H), 1.72 (s, 3H), 1.57-1.59 (m, 4H), 1.10 (s,
9H)
[0609] .sup.13C NMR (101 MHz, Chloroform-d): .delta. 163.4, 150.2,
144.6, 135.6, 135.2, 134.9, 132.8, 131.7, 130.2, 130.1, 128.1,
128.0, 112.2, 111.6, 86.0, 85.9 (d, J=2.9 Hz), 84.4, 79.6 (d, J=7.3
Hz), 65.7, 63.8, 39.2 (d, J=8.0 Hz), 38.8, 33.7, 33.6, 27.8, 27.6,
27.0, 23.4, 22.6, 21.7, 19.4, 11.9;
[0610] .sup.31P NMR (162 MHz, Chloroform-d): .delta. 101.49;
[0611] HRMS (ESI-TOF, m/z): Calcd for
[C.sub.36H.sub.47N.sub.2O.sub.6PS.sub.2Si+H].sup.+ 727.2455; Found
727.2478 (3.1 ppm error).
[0612] R.sub.f=0.41 (40% EtOAc in hexane); UV, KMnO.sub.4.
2.6. Compound 3-112
##STR00448##
[0614] A suspension of nucleoside
1-((2R,4S,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-hydroxytetrahydr-
ofuran-2-yl)-5-methylpyrimidine-2.4(1H,3H)-dione (intermediate
16-iv) (3.00 g, 6.24 mmol, 1.0 equiv.) and the (-)-.psi. reagent
(3.62 g, 8.11 mmol, 1.3 equiv.) in MeCN (62 mL) was cooled to
0.degree. C. DBU (1.22 mL, 8.11 mmol, 1.3 equiv.) was added in one
portion, stirred at 0.degree. C. for 30 min then the mixture, then
passed through a plug of silica gel (ca. 1'') and washed with ethyl
acetate (62 mL). The solution was washed with water (31 mL) then
with K.sub.2HPO.sub.4 (10 wt % aq., 31 mL). The organic layer was
dried over MgSO.sub.4, filtered and concentrated in vacuo to afford
a yellow gel that was purified by flash column chromatography (10%
to 90% EtOAc in hexanes) to afford a foam which was broken down
into a powder by concentration from heptane (3.times.50 mL). The
compound 3-112 was isolated as a white powder which was dried at
50.degree. C. and 20 Torr until constant weight was reached (4.46
g, 91.5 wt %, remainder heptane, 90% corrected yield).
[0615] Physical State: White solid;
[0616] .sup.1H NMR (400 MHz, Chloroform-d): .delta. 8.22 (br s,
1H), 7.64-7.74 (m, 4H), 7.51 (d, J=1.3 Hz, 1H), 7.38-7.50 (m, 6H),
6.45 (dd, J=9.3, 5.1 Hz, 1H), 5.59 (dd, J=11.4, 5.8 Hz, 1H), 5.09
(s, 1H), 4.93 (s, 1H), 4.50 (dt, J=12.7, 3.3 Hz, 1H), 4.24 (d,
J=1.5 Hz, 1H), 3.97 (d, J=2.0 Hz, 2H), 2.57-2.65 (br m, 1H), 2.57
(dd, J=14.0, 5.4 Hz, 1H), 2.27-2.38 (m, 2H), 2.14 (td, J=13.3, 3.9
Hz, 1H), 1.86-2.02 (m, 3H), 1.82 (s, 3H), 1.72-1.82 (m, 1H), 1.72
(s, 3H), 1.59 (d, J=1.0 Hz, 3H), 1.10 (s, 9H);
[0617] .sup.13C NMR (101 MHz, Chloroform-d): .delta. 163.6, 150.2,
144.6, 135.6, 135.2, 134.9, 132.8, 131.7, 130.2, 130.0, 128.1,
128.0, 112.2, 111.5, 86.0, 85.8 (d, J=7.1 Hz), 84.2, 79.1 (d, J=8.1
Hz), 65.8, 63.9, 39.5 (d, J=4.0 Hz), 38.8, 33.7, 33.6, 27.8, 27.6,
27.0, 23.3, 22.6, 21.7, 19.3, 11.9;
[0618] .sup.31P NMR (162 MHz, Chloroform-d): .delta. 101.67;
[0619] HRMS (ESI-TOF, m/z): HRMS (ESI) Calcd for
[C.sub.36H.sub.47N.sub.2O.sub.6PS.sub.2Si+H].sup.+ 727.2455; Found
727.2474 (2.6 ppm error).
[0620] R.sub.f=0.41 (40% EtOAc in hexane); UV, KMnO.sub.4.
2.7. Compound 3-113
##STR00449##
[0622] To a 100 mL flask were added
(E)-N'-(9-((2R,4S,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-hydroxyt-
etrahydrofuran-2-yl)-6-hydroxy-9H-purin-2-yl)-N,N-dimethylformimidamide
(intermediate 16-iii) (1.50 g, 2.60 mmol, 1.0 equiv.) and (-)-.psi.
(1.67 g, 3.74 mmol, 1.4 equiv.) in MeCN (27 mL). The mixture was
cooled to 0.degree. C. and DBU (0.52 mL, 3.48 mmol, 1.3 equiv.) was
added dropwise; the reaction was left to stir at ambient
temperature. After 1 h UPLC analysis showed complete consumption of
intermediate 16-iii. The reaction mixture was diluted with EtOAc
(27 mL) then washed with water (27 mL) and Na.sub.2HPO.sub.4 (10 wt
%, 27 mL). The organic phase was dried over Na.sub.2SO.sub.4,
filtered and the solvent was removed in vacuo. The crude residue
was purified by silica gel column chromatography (100% EtOAc to
100% THF) to afford the compound 3-113 as a light yellow solid
(1.96 g, 91%). Note: The isolated product contains BHT (from
stabilized THF).
[0623] Physical State: White solid;
[0624] .sup.1H NMR (400 MHz, Chloroform-d): .delta. 9.90 (br s,
1H), 8.52 (s, 1H), 7.79 (s, 1H), 7.69-7.60 (m, 4H), 7.44-7.28 (m,
6H), 6.32 (dd, J=7.7, 6.2 Hz, 1H), 5.64 (ddt, J=11.5, 6.0, 2.7 Hz,
1H), 4.99 (s, 1H), 4.87 (s, 1H), 4.47 (dt, J=12.7, 3.1 Hz, 1H),
4.28 (q, J=3.4 Hz, 1H), 3.92-3.82 (m, 2H), 3.12 (s, 3H), 3.06 (s,
3H), 2.90-2.55 (m, 4H), 2.27-2.22 (m, 1H), 2.18-2.04 (m, 1H),
2.00-1.80 (m, 3H), 1.76 (s, 3H), 1.73 (s, 3H), 1.04 (s, 9H);
[0625] .sup.13C NMR (101 MHz, Chloroform-d): .delta. 158.4, 157.8,
156.8, 150.0, 145.0, 136.4, 135.5, 135.4, 132.5, 132.3, 129.9,
127.8, 127.8, 120.5, 111.8, 86.0, 85.8, 85.7, 83.4, 79.0, 78.9,
65.8, 63.5, 41.3, 39.0, 39.0, 38.7, 35.2, 33.7, 33.6, 27.7, 27.5,
26.8, 23.3, 22.6, 21.7, 19.1;
[0626] .sup.31P NMR (162 MHz, Chloroform-d): .delta. 100.5;
[0627] HRMS (ESI-TOF, m/z): HRMS (ESI) Calcd for
[C.sub.39H.sub.51N.sub.6O.sub.5PS.sub.2Si+H].sup.+807.2942; Found
807.2957 (1.8 ppm error).
[0628] R.sub.f=0.68 (100% THF); UV, KMnO.sub.4.
2.8. Compound 3-114
##STR00450##
[0630] To a 100 mL flask were added
(E)-N'-(9-((2R,4S,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-4-hydroxyt-
etrahydrofuran-2-yl)-6-hydroxy-9H-purin-2-yl)-N,N-dimethylformimidamide
(intermediate 16-iii) (1.10 g, 1.96 mmol, 1.0 equiv.) and the
(-)-.psi. reagent (1.14 g, 2.55 mmol, 1.4 equiv.) in MeCN (20 mL).
The mixture was cooled to 0.degree. C. and DBU (0.38 mL, 2.55 mmol,
1.3 equiv.) was added dropwise and the reaction was left to stir at
ambient temperature. After 1 h UPLC analysis showed complete
consumption of SI-3. The reaction mixture was diluted with EtOAc
(20 mL) then washed with water (20 mL) and Na.sub.2HPO.sub.4 (10 wt
%, 20 mL). The organic phase was dried over Na.sub.2SO.sub.4,
filtered, and the solvent was removed in vacuo. The crude residue
was purified by silica gel column chromatography (0 to 100% THF in
EtOAc) to afford the compound 3-114 as a white solid (1.20 g, 76%).
Note: The isolated product contains BHT (from stabilized THF).
[0631] Physical State: White solid;
[0632] .sup.1H NMR (400 MHz, Chloroform-d): .delta. 9.61 (br s,
1H), 8.57 (s, 1H), 7.77 (s, 1H), 7.69-7.60 (m, 4H), 7.44-7.32 (m,
6H), 6.32 (dd, J=7.7, 6.2 Hz, 1H), 5.64 (ddt, J=11.5, 6.0, 2.7 Hz,
1H), 5.00 (s, 1H), 4.88 (s, 1H), 4.48 (dt, J=12.7, 3.1 Hz, 1H),
4.27 (q, J=3.4 Hz, 1H), 3.92-3.82 (m, 2H), 3.14 (s, 3H), 3.10 (s,
3H), 2.93-2.81 (m, 1H), 2.71-2.58 (m, 2H), 2.37-2.27 (m, 1H),
2.18-2.04 (m, 1H), 2.00-1.80 (m, 4H), 1.77 (s, 3H), 1.73 (s, 3H),
1.05 (s, 9H);
[0633] .sup.13C NMR (101 MHz, Chloroform-d): .delta. 158.0, 157.8,
156.8, 150.0, 145.0, 136.2, 135.6, 135.4, 132.6, 132.4, 129.9,
127.8, 127.8, 120.8, 111.8, 85.9, 85.6, 85.5, 83.2, 78.5, 78.5,
65.8, 63.5, 41.3, 39.0, 39.0, 38.8, 35.2, 33.8, 33.7, 27.7, 27.6,
26.9, 23.3, 22.7, 21.7, 19.1;
[0634] .sup.31P NMR (162 MHz, Chloroform-d): .delta. 100.9;
[0635] HRMS (ESI-TOF, m/z): HRMS (ESI) Calcd for
[C.sub.3H.sub.51N.sub.6O.sub.5PS.sub.2Si+H].sup.+807.2942; Found
807.2957 (1.8 ppm error).
[0636] R.sub.f=0.68 (100% THF); UV, KMnO.sub.4.
2.9. Compound 3-115
##STR00451##
[0638] Compound 3-115 was prepared according to General Procedure 2
using 5'-O-(4,4'-dimethoxytrityl)-2'-deoxythymidine (544 mg, 1.00
mmol). Purification by silica gel column chromatography (30 to 50%
EtOAc in hexanes with 1% Et.sub.3N) afforded compound 3-115 (459
mg, 58%).
[0639] Physical State: White solid;
[0640] .sup.1H NMR (600 MHz, Acetone-d.sub.6): .delta. 10.04 (s,
1H), 7.62 (d, J=1.5 Hz, 1H), 7.54-7.49 (m, 2H), 7.42-7.31 (m, 6H),
7.30-7.24 (m, 2H), 6.96-6.90 (m, 4H), 6.38 (dd. J=8.2, 6.1 Hz, 1H),
5.58 (ddt, J=10.8, 5.5, 2.4 Hz, 1H), 5.02 (q, J=1.5 Hz, 1H),
4.97-4.93 (m, 1H), 4.53 (dt, J=12.8, 3.4 Hz, 1H), 4.26 (q, J=3.0
Hz, 1H), 3.81 (s, 6H), 3.50 (dd, J=10.6, 3.4 Hz, 1H), 3.41 (dd,
J=10.6, 3.2 Hz, 1H), 2.67 (s, 1H), 2.70-2.57 (m, 2H), 2.35 (ddd,
J=13.2, 3.6, 1.8 Hz, 1H), 2.12-2.04 (m, 1H), 2.03-1.94 (m, 3H),
1.89 (ddt, J=13.9, 10.5, 5.6 Hz, 1H), 1.81 (s, 3H), 1.71 (s, 3H),
1.49 (d, J=1.2 Hz, 3H).
[0641] .sup.13C NMR (151 MHz, Acetone): .delta. 164.11, 159.79,
159.77, 151.20, 146.30, 145.70, 136.45, 136.25, 136.01, 131.00,
130.99, 128.96, 128.79, 127.80, 114.08, 112.18, 111.32, 87.77,
86.78, 85.10, 85.05, 85.04, 80.01, 79.96, 66.91, 64.22, 55.55,
39.77, 39.48, 39.46, 34.47, 34.41, 28.26, 28.15, 23.93, 22.79,
22.06, 12.15.
[0642] .sup.31P NMR (162 MHz, Acetone): .delta. 101.75.
[0643] HRMS (ESI-TOF, m/z): Calcd for
C.sub.41H.sub.47N.sub.2O.sub.8PS.sub.2 [M-DMTr+H].sup.+489.1277.;
found 489.1278.
[0644] R.sub.f=0.43 (5% Acetone in DCM); UV, KMnO.sub.4.
2.10. Compound 3-116
##STR00452##
[0646] Compound 3-116 prepared according to General Procedure 2
using 5'-O-(tert-butyldimethylsilyl)-2'-deoxy-2'-fluoroadenosine
Intermediate 16-v (1.37 g, 3.58 mmol). Crystallization of the crude
reaction mixture from acetonitrile afforded compound 3-116 (1.85 g,
82%).
[0647] Physical State: White solid;
[0648] .sup.1H NMR (600 MHz, Acetone-d.sub.6): .delta. 8.22 (d,
J=7.6 Hz, 2H), 6.71 (s, 2H), 6.35 (dd, J=17.8, 2.1 Hz, 1H),
5.89-5.74 (m, 2H), 5.02 (q, J=1.5 Hz, 1H), 4.97-4.93 (m, 1H), 4.59
(dt, J=12.8, 3.5 Hz, 1H), 4.33 (dt, J=6.2, 3.1 Hz, 1H), 4.06 (dd,
J=11.8, 2.8 Hz, 1H), 3.90 (dd, J=11.8, 3.4 Hz, 1H), 2.86 (s, 1H),
2.65 (d, J=5.8 Hz, 1H), 2.30 (dtd, J=13.4, 3.3, 1.6 Hz, 1H), 2.12
(td, J=13.6, 4.4 Hz, 1H), 2.05-1.83 (m, 4H), 1.81-1.78 (m, 3H),
1.70 (s, 3H), 0.89 (s, 9H), 0.07 (d, J=22.5 Hz, 6H).
[0649] .sup.13C NMR (151 MHz, Acetone-d.sub.6): .delta. 157.23,
153.94, 150.28, 146.20, 140.03, 120.64, 112.05, 93.35, 92.10,
87.64, 87.42, 86.66, 82.85, 82.79, 73.87, 73.83, 73.78, 73.73,
67.33, 62.14, 39.79, 34.41, 34.35, 28.23, 28.13, 26.35, 23.93,
22.67, 22.09, 18.95, -5.20, -5.30.
[0650] .sup.19F NMR (376 MHz, Acetone-d.sub.6): .delta.
-202.95.
[0651] .sup.31P NMR (162 MHz, Acetone-d.sub.6): .delta. 101.65.
[0652] HRMS (ESI-TOF, m/z): Calcd for
C.sub.2H.sub.41FN.sub.5O.sub.4PS.sub.2Si [M+H].sup.+ 630.2164.;
found 630.2167.
[0653] R.sub.f=0.43 (20% acetone in DCM); UV, KMnO.sub.4.
2.11. Compound 3-117
##STR00453##
[0655] Compound 3-117 was prepared according to General Procedure 2
using 5'-O-(tert-butyldimethylsilyl)-2'-deoxy-2'-fluoroadenosine
intermediate 16-v (685 mg, 1.79 mmol). Crystallization of the crude
reaction mixture from acetonitrile afforded compound 3-117 (933 mg,
83%).
[0656] Physical State: White solid;
[0657] .sup.1H NMR (600 MHz, CDCl.sub.3): .delta. 8.39 (s, 1H),
8.16 (s, 1H), 6.39 (dd, J=14.3, 3.7 Hz, 1H), 5.82 (s, 2H),
5.65-5.49 (m, 2H), 5.06 (q, J=1.4 Hz, 1H), 4.94-4.90 (m, 1H), 4.55
(ddd, J=12.8, 3.7, 2.5 Hz, 1H), 4.44 (h, J=2.1 Hz, 1H), 4.04 (dd,
J=11.7, 2.3 Hz, 1H), 3.90 (dd, J=11.7, 2.7 Hz, 1H), 2.62 (s, 1H),
2.33 (ddt, J=13.0, 3.8, 1.7 Hz, 1H), 2.18 (td, J=13.5, 4.2 Hz, 1H),
2.04-1.86 (m, 3H), 1.74 (s, 3H), 0.95 (s, 7H), 0.14 (d, J=8.8 Hz,
7H);
[0658] .sup.13C NMR (151 MHz. CDCl.sub.3): .delta. 155.00, 152.90,
149.28, 144.34, 138.10, 119.43, 111.59, 91.66, 91.64, 90.35, 90.33,
85.84, 85.50, 85.29, 82.70, 82.66, 73.80, 73.75, 73.70, 73.66,
65.18, 61.27, 38.41, 33.23, 33.17, 27.33, 27.22, 25.53, 22.92,
22.23, 21.24, 17.99, -5.82, -5.86;
[0659] .sup.19F NMR (376 MHz, CDCl.sub.3): .delta. -204.21;
[0660] .sup.31P NMR (162 MHz, CDCl.sub.3): .delta. 103.12;
[0661] HRMS (ESI-TOF, m/z): Calcd for
C.sub.26H.sub.41FN.sub.5O.sub.4PS.sub.2Si [M+H].sup.+ 630.2164.;
found 630.2165.
[0662] R.sub.f=0.43 (20% acetone in DCM); UV, KMnO.sub.4.
2.12. Compound 3-118
##STR00454##
[0664] Compound 3-118 was prepared according to General Procedure 2
using N.sup.6-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine
(657 mg, 1.00 mmol). Purification by silica gel column
chromatography (30 to 50% EtOAc in hexanes with 1% TEA) afforded
compound 3-118 (461 mg, 51%).
[0665] Physical State: White solid;
[0666] .sup.1H NMR (600 MHz, Acetone-d.sub.6): .delta. 10.00 (s,
1H), 8.58 (s, 1H), 8.42 (s, 1H), 8.14 (d, J=7.4 Hz, 2H), 7.69-7.62
(m, 1H), 7.57 (t, J=7.8 Hz, 2H), 7.54-7.46 (m, 2H), 7.38-7.26 (m,
7H), 7.25-7.19 (m, 1H), 6.89-6.82 (m, 4H), 6.58 (dd, J=7.7, 6.1 Hz,
1H), 5.71 (ddt, J=11.3, 5.7, 2.7 Hz, 1H), 5.05 (q, J=1.5 Hz, 1H),
4.98 (d, J=1.8 Hz, 1H), 4.58 (dt, J=12.8, 3.4 Hz, 1H), 4.39 (td,
J=5.0, 2.6 Hz, 1H), 3.79 (d, J=2.5 Hz, 6H), 3.52-3.35 (m, 3H),
2.87-2.78 (m, 1H), 2.68 (s, 1H), 2.36 (ddq, J=13.8, 3.3, 1.6 Hz,
1H), 2.12 (td, J=13.6, 4.5 Hz, 1H), 2.07-1.96 (m, 2H), 1.95-1.85
(m, 1H), 1.82 (s, 3H), 1.73 (s, 3H).
[0667] .sup.13C NMR (151 MHz, Acetone-d.sub.6): .delta. 159.75,
152.98, 152.70, 151.45, 146.60, 146.05, 143.56, 136.73, 135.21,
133.87, 133.35, 131.13, 131.07, 130.56, 129.54, 129.30, 129.11,
128.74, 127.72, 126.37, 114.06, 112.29, 87.41, 87.01, 85.63, 85.58,
85.56, 80.20, 80.15, 67.03, 64.30, 55.66, 39.95, 38.49, 38.47,
34.72, 34.66, 28.45, 28.34, 24.11, 22.99, 22.24.
[0668] .sup.31P NMR (162 MHz, Acetone): .delta. 101.42.
[0669] HRMS (ESI-TOF, m/z): Calcd for
C.sub.48H.sub.50N.sub.5O.sub.7PS.sub.2 [M+H].sup.+ 904.2963.; found
904.2968.
[0670] R.sub.f=0.57 (5% Acetone in DCM); UV, KMnO.sub.4.
2.13. Compound 3-119
##STR00455##
[0672] Compound 3-119 was prepared according to General Procedure 2
using N.sup.4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine
(634 mg, 1.00 mmol). Purification by silica gel column
chromatography (30 to 50% EtOAc in hexanes with 1% Et.sub.3N)
afforded 3-119 (395 mg, 45%).
[0673] Physical State: White solid;
[0674] .sup.1H NMR (600 MHz, CDCl.sub.3): .delta. 8.68 (s, 1H),
8.20 (d, J=7.5 Hz, 1H), 7.91 (d, J=7.7 Hz, 2H), 7.63 (t, J=7.5 Hz,
1H), 7.54 (t, J=7.5 Hz, 2H), 7.41 (d, J=7.2 Hz, 2H), 7.36-7.29 (m,
6H), 7.28-7.24 (m, 1H), 6.88 (dd, J=8.8, 3.5 Hz, 4H), 6.37 (t,
J=6.5 Hz, 1H), 5.61-5.52 (m, 1H), 5.09 (s, 1H), 4.94 (s, 1H), 4.48
(dt, J=12.7, 3.2 Hz, 1H), 4.43-4.39 (m, 1H), 3.82 (s, 3H), 3.81 (s,
3H), 3.55 (dd, J=10.9, 3.3 Hz, 1H), 3.46 (dd, J=10.9, 2.8 Hz, 1H),
2.93 (ddd, J=14.4, 5.8, 2.8 Hz, 1H), 2.62 (d, J=6.2 Hz, 1H),
2.46-2.33 (m, 2H), 2.15 (td, J=13.5, 4.2 Hz, 1H), 2.03-1.97 (m,
1H), 1.96-1.88 (m, 2H), 1.85 (s, 3H), 1.82-1.75 (m, 1H), 1.73 (s,
3H) ppm;
[0675] .sup.13C NMR (151 MHz, CDCl.sub.3): .delta. 162.2, 158.8,
158.8, 144.8, 144.1, 135.3, 135.1, 133.3, 130.2, 130.1, 129.2,
128.2, 127.6, 127.3, 113.5, 113.5, 112.3, 87.3, 87.2, 86.2, 85.8,
85.7, 78.6, 78.6, 66.1, 62.8, 55.4, 55.4, 40.9, 40.9, 39.0, 33.9,
33.8, 27.9, 27.8, 25.7, 23.5, 22.9, 21.9 ppm;
[0676] .sup.31P NMR (162 MHz, CDCl.sub.3): .delta. 102.1 ppm;
[0677] HRMS (ESI-TOF): calcd. for
C.sub.47H.sub.51N.sub.3O.sub.8PS.sub.2 [M+H].sup.+ 880.2855; found
880.2878.
[0678] R.sub.f=0.25 (30% EtOAc in hexanes); UV, KMnO.sub.4.
2.14. Compound 3-120
##STR00456##
[0680] Compound 3-120 was prepared according to General Procedure 2
using N.sup.6-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine
(657 mg, 1.00 mmol). Purification by silica gel column
chromatography (50 to 100% EtOAc in hexanes with 0.1% TEA) afforded
compound 3-120 (263 mg, 30%).
[0681] Physical State: White solid;
[0682] .sup.1H NMR (600 MHz, Acetone-d.sub.6): .delta. 10.26 (s,
1H), 7.94 (s, 1H), 7.47-7.42 (m, 2H), 7.34-7.29 (m, 4H), 7.26 (s,
1H), 7.26-7.17 (m, 2H), 6.87-6.80 (m, 4H), 6.26 (dd, J=8.2, 5.8 Hz,
1H), 5.55 (ddt, J=11.3, 5.5, 2.6 Hz, 1H), 5.00 (q, J=1.4 Hz, 1H),
4.93 (dt, J=1.9, 0.9 Hz, 1H), 4.50 (dt, J=12.8, 3.4 Hz, 1H), 4.25
(td, J=4.6, 2.5 Hz, 1H), 4.05 (q, J=7.2 Hz, 1H), 3.77 (s, 6H), 3.44
(dd, J=10.5, 5.1 Hz, 1H), 3.34 (dd, J=10.4, 4.2 Hz, 1H), 3.15 (ddd,
J=14.1, 8.2, 5.9 Hz, 1H), 2.72 (ddd, J=14.1, 5.9, 2.6 Hz, 1H), 2.66
(s, 1H), 2.36-2.29 (m, 1H), 2.12-1.94 (m, 9H), 1.88 (ddt, J=15.0,
13.2, 4.9 Hz, 1H), 1.77 (dt, J=1.4, 0.7 Hz, 3H), 1.71 (s, 3H),
1.25-1.17 (m, 8H).
[0683] .sup.13C NMR (151 MHz, Acetone): .delta. 180.74, 180.67,
171.03, 159.80, 159.79, 155.85, 149.55, 149.24, 146.62, 146.01,
137.95, 136.72, 136.65, 131.14, 131.08, 129.12, 128.75, 127.77,
122.46, 114.06, 112.27, 87.44, 87.12, 85.73, 85.68, 84.65, 80.05,
80.00, 67.12, 64.45, 60.68, 55.65, 39.92, 38.70, 38.67, 36.80,
36.76, 34.73, 34.67, 30.50, 30.34, 30.21, 29.85, 29.70, 28.44,
28.34, 24.07, 22.94, 22.21, 20.98, 19.50, 19.36, 14.65.
[0684] .sup.31P NMR (162 MHz, Acetone): .delta. 100.84.
[0685] HRMS (ESI) m/z: calculated for
C.sub.45H.sub.52N.sub.5O.sub.8PS.sub.2 [M+H].sup.+ 886.3068; found
886.3066.
[0686] R.sub.f=0.25 (60% EtOAc in hexanes+0.1% Et.sub.3N); UV,
KMnO.sub.4.
2.15. Compound 3-121
##STR00457##
[0688] Compound 3-121 was prepared according to General Procedure 2
using 5'-O-(tert-butyldimethylsilyl)-2'-deoxyadenosine (585 mg, 1.6
mmol). Purification by flash column chromatography (70% EtOAc in
hexanes) afforded compound 3-121 (628 mg, 64% yield).
[0689] Physical State: White solid;
[0690] .sup.1H NMR (600 MHz, Acetone-d.sub.6): .delta. 8.21 (d,
J=7.5 Hz, 2H), 6.61 (s, 1H), 6.49-6.41 (m, 1H), 5.54 (ddt, J=10.4,
5.6, 2.2 Hz, 1H), 5.06 (q, J=1.5 Hz, 1H), 5.01-4.98 (m, 1H), 4.57,
(dt, J=12.8, 3.4 Hz, 1H), 4.26 (td, J=4.5, 2.0 Hz, 1H), 3.98 (dd,
J=11.1, 5.0 Hz, 1H), 3.89 (dd, J=11.2, 4.0 Hz, 1H), 3.11 (ddd,
J=14.0, 8.3, 5.7 Hz, 1H), 2.74 (ddd, J=14.2, 5.9, 2.3 Hz, 1H), 2.68
(s, 1H), 2.37 (ddt, J=13.4, 3.3, 1.7 Hz, 1H), 2.13 (td, J=13.6, 4.4
Hz, 1H), 2.05-1.95 (m, 5H), 1.89 (tdd, J=14.8, 6.0, 4.5 Hz, 1H),
1.82 (s, 3H), 1.71 (s, 3H), 0.95 (s, 1H), 0.93 (s, 9H), 0.17 (d,
J=2.7 Hz, 1H), 0.12 (s, 6H), 0.08 (d, J=8.0 Hz, 1H).
[0691] .sup.13C NMR (151 MHz, Acetone): .delta. 157.31, 153.89,
150.76, 146.59, 139.92, 112.34, 87.03, 86.70, 85.04, 80.47, 73.55,
67.02, 64.06, 39.98, 39.35, 34.64, 28.36, 26.53, 24.12, 22.99,
22.23, 19.10, -5.04.
[0692] .sup.31P NMR (162 MHz, Acetone): .delta. 100.35.
[0693] HRMS (ESI) m/z: calculated for
C.sub.26H.sub.42NSO.sub.4PS.sub.2Si [M+H].sup.+ 612.2258; found
612.2258.
[0694] R.sub.f=0.31 (60% EtOAc in hexanes+0.1% Et.sub.3N); UV,
KMnO.sub.4.
2.16. Compound 3-122
##STR00458##
[0696] Compound 3-122 was prepared according to General Procedure 2
using 5'-O-(tert-butyldimethylsilyl)-2'-deoxyadenosine (516 mg,
0.93 mmol). Purification by flash column chromatography (20%
Acetone in DCM) afforded compound 3-122 (454 mg, 58% yield).
[0697] Physical State: White solid;
[0698] .sup.1H NMR (600 MHz, Acetone-d.sub.6): .delta. 8.13 (d,
J=4.4 Hz, 2H), 7.50-7.44 (m, 2H), 7.37-7.31 (m, 4H), 7.27 (t, J=7.7
Hz, 2H), 7.24-7.16 (m, 1H), 6.87-6.80 (m, 4H), 6.67 (s, 2H), 6.44
(dd, J=7.9, 6.1 Hz, 1H), 5.65 (ddt, J=11.2, 5.6, 2.6 Hz, 1H), 5.03
(q, J=1.5 Hz, 1H), 4.97-4.94 (m, 1H), 4.54 (dt, J=12.8, 3.4 Hz,
1H), 4.32 (td, J=5.0, 2.5 Hz, 1H), 3.77 (d, J=1.5 Hz, 6H), 3.47
(dd, J=10.3, 5.2 Hz, 1H), 3.39 (dd, J=10.3, 5.0 Hz, 1H), 3.32 (ddd,
J=14.0, 7.9, 5.9 Hz, 1H), 2.72 (ddd, J=14.2, 6.1, 2.7 Hz, 1H), 2.66
(s, 1H), 2.34 (ddq, J=13.4, 3.1, 1.6 Hz, 1H), 2.09 (s, 1H),
2.04-1.96 (m, 3H), 1.88 (dddd, J=14.9, 13.5, 6.0, 4.5 Hz, 1H), 1.80
(s, 3H), 1.70 (s, 3H).
[0699] .sup.13C NMR (151 MHz, Acetone-d.sub.6): .delta. 158.73,
158.71, 156.23, 152.72, 149.65, 145.54, 145.06, 139.47, 135.74,
130.11, 130.06, 128.10, 127.70, 126.67, 119.99, 113.01, 111.27,
86.36, 85.94, 84.42, 84.36, 84.24, 79.34, 79.29, 65.97, 63.30,
54.61, 38.91, 37.48, 37.46, 33.68, 33.62, 29.72, 27.40, 27.30,
23.07, 21.95, 21.20.
[0700] .sup.31P NMR (162 MHz, Acetone-d.sub.6): .delta. 100.33.
[0701] HRMS (ESI) m/z: calculated for
C.sub.41H.sub.46N.sub.5O.sub.6PS.sub.2 [M+H].sup.+ 800.2700; found
800.2701.
[0702] R.sub.f=0.29 (20% Acetone in DCM
2.17. Compound 3-128
##STR00459##
[0704] A dry 250 mL round bottom flask was charged with
(2R,3aR,6S,7aR)-3a-methyl-2-((perfluorophenyl)thio)-6-(prop-1-en-2-yl)hex-
ahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide, (4.40 g, 9.86
mmol) and N6-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine
(5.0 g, 7.60 mmol) under a stream of nitrogen.
N,N-Dimethylformamide (30 mL) was added to the mixture which was
allowed to stir at room temperature until it became homogeneous.
The mixture was cooled to 0.degree. C., and
1,8-diazabicyclo[5.4.0]undec-7-ene (1.5 mL, 10.03 mmol) was then
added drop-wise over 1 minute. The reaction mixture was allowed to
stir at 0.degree. C. for a total of 15 minutes. The cold reaction
mixture was diluted with toluene (150 mL). The mixture was
transferred to a separatory funnel where it was washed twice with
1M pH 7.4 phosphate buffer solution (Aldrich P3619, 100 mL). The
organics were dried using MgSO.sub.4 and the mixture was filtered
and the filtrate was concentrated to dryness. The residue was
dissolved in ethyl acetate (<5 mL). The mixture was purified via
Biotage medium pressure liquid chromatography (Silica; Isco RediSep
120 g; 70% EtOAc-Hex 1% triethylamine to 100% Ethyl acetate 1%
triethylamine over 16 CV), affording 4.24 g of solid white foam.
LCMS (Waters Aquity UPLC BEH C18 2.1.times.50 mm 1.7 mm; 2%
CH3CN--H2O 0.05% TFA to 98% CH3CN--H2O 0.05% TFA over 1.5 min at
0.8 mL/min, 50.degree. C.) (M+H)*=904.5 (rf 1.66 min). 1H NMR (500
MHz, CHLOROFORM-d) .delta. 9.03 (s, 1H), 8.76 (s, 1H), 8.20 (s,
1H), 8.08-7.98 (m, 2H), 7.67-7.60 (m, 1H), 7.57-7.51 (m, 2H),
7.45-7.40 (m, 2H), 7.35-7.30 (m, 4H), 7.30-7.25 (m, 2H), 7.25-7.19
(m, 1H), 6.82 (d, J=9.0 Hz, 4H), 6.58 (dd, J=8.8, 5.6 Hz, 1H), 5.67
(br dd, J=11.1, 5.4 Hz, 1H), 5.06 (s, 1H), 4.92 (s, 1H), 3.79 (s,
5H), 3.56-3.38 (m, 2H), 3.11-2.98 (m, 1H), 2.83 (dd, J=13.9, 5.6
Hz, 1H), 2.62 (br s, 1H), 2.29 (br d, J=13.1 Hz, 1H), 2.17 (td,
J=13.4, 3.9 Hz, 1H), 2.04-1.86 (m, 4H), 1.80 (s, 3H), 1.85-1.76 (m,
2H), 1.74 (s, 3H).
2.18. Compound 3-129
##STR00460##
[0706] A dry 250 mL round bottom flask was charged with
(2R,3aR,6S,7aR)-3a-methyl-2-((perfluorophenyl)thio)-6-(prop-1-en-2-yl)hex-
ahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide (4.45 g, 9.97 mmol)
and
5'-O-(4,4'-dimethoxytrityl)-n4-benzoyl-5-methyl-2'-deoxycytidine
(5.00 g, 7.72 mmol) under a stream of nitrogen.
N,N-Dimethylformamide (4 mL) was added to the mixture which was
allowed to stir at room temperature until it became homogeneous.
The mixture was cooled to 0.degree. C.
1,8-diazabicyclo[5.4.0]undec-7-ene (1.5 mL, 10.03 mmol) was then
added to the mixture drop-wise. The mixture was stirred at
0.degree. C. for 20 minutes. The cold reaction mixture was diluted
to -25 mL with toluene. The mixture was transferred to a separatory
funnel where it was washed twice with 1M pH 7.4 phosphate buffer
solution (Aldrich P3619). Organics were dried using MgSO.sub.4. The
mixture was filtered and the filtrate was concentrated to dryness.
The residue was dissolved in ethyl acetate (<5 mL). The mixture
was purified via Biotage medium pressure liquid chromatography
(Silica; Isco RediSep 24 g; 20% EtOAc-Hex 1% triethylamine to 50%
EtOAc-Hex 1% triethylamine over 16 CV). Major peak was isolated and
concentrated to dryness. The resulting residue was treated with
acetonitrile (50 mL). The mixture was then filtered through a 0.45
m PVDF Whatman syringe filter. The filtrate was then concentrated
to dryness, 4.95 g of solid white foam was obtained. 1H NMR (500
MHz, CHLOROFORM-d) .delta. 13.30 (br s, 1H), 8.32 (d, J=7.5 Hz,
2H), 7.82 (s, 1H), 7.60-7.51 (m, 1H), 7.48-7.40 (m, 4H), 7.37-7.30
(m, 6H), 7.27-7.22 (m, 1H), 6.87 (d, J=8.7 Hz, 4H), 6.53 (dd,
J=8.9, 5.4 Hz, 1H), 5.65 (dd, J=11.1, 6.0 Hz, 1H), 5.07 (s, 1H),
4.90 (s, 1H), 4.47 (dt, J=12.7, 3.0 Hz, 1H), 4.34 (s, 1H), 3.81 (s,
6H), 3.63-3.38 (m, 2H), 2.69 (dd, J=14.0, 5.5 Hz, 1H), 2.60 (br s,
1H), 2.52-2.33 (m, 2H), 2.25 (br d, J=1.9 Hz, 1H), 2.17 (td,
J=13.5, 4.0 Hz, 1H), 2.04-1.91 (m, 2H), 1.86 (td, J=13.1, 5.9 Hz,
1H), 1.77-1.72 (m, 1H), 1.71 (s, 3H), 1.69-1.64 (m, 2H), 1.63 (s,
3H), 31P NMR (202 MHz, CHLOROFORM-d) .delta. 101.4 (br d, J=10.0
Hz, 1P).
[0707] LCMS (Waters Aquity UPLC BEH C18 2.1.times.50 mm 1.7 mm; 2%
CH3CN--H2O 0.05% TFA to 98% CH3CN--H2O 0.05% TFA over 1.5 min at
0.8 mL/min) (M+H)+=894.5 (rf 1.93 min). Data are consistent with
the desired
N-(1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((2S,-
3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2-yl)-2-sulfidohexahydrobenzo[d][1,3,2]-
oxathiaphosphol-2-yl)oxy)tetrahydrofuran-2-yl)-5-methyl-2-oxo-1,2-dihydrop-
yrimidin-4-yl)benzamide (4.95 g, 5.54 mmol, 71.7% yield).
2.19. Compound 3-130
##STR00461##
[0709] A dry 250 mL round bottom flask was charged with
(2R,3aR,6S,7aR)-3a-methyl-2-((perfluorophenyl)thio)-6-(prop-1-en-2-yl)hex-
ahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide (5.8 g, 12.99 mmol)
and
N-(9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydrox-
ytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide
(8.9 g, 13.9 mmol) under a stream of nitrogen.
N,N-Dimethylformamide (50 mL) was added to the mixture which was
allowed to stir at room temperature until it became homogeneous.
The mixture was cooled to 0.degree. C.
1,8-diazabicyclo[5.4.0]undec-7-ene (2.081 mL, 13.91 mmol) was then
added to the mixture drop-wise. The mixture was stirred at
0.degree. C. for 10 minutes.
[0710] The cold reaction mixture was diluted with .about.200 mL
toluene. The mixture was transferred to a separatory funnel where
it was washed twice with 1M pH 7.4 phosphate buffer solution
(Aldrich P3619). Organics were dried with Na.sub.2SO.sub.4. The
mixture was filtered and the filtrate was concentrated to dryness.
9.25 g. Evaporate onto celite (toluene) The mixture was purified
via Biotage medium pressure liquid chromatography (Silica; Isco
RediSep 120 g; 70% EtOAc-Hex 1% triethylamine to 100% Ethyl acetate
1% triethylamine over 6 CV, hold at 100% for 10CV). Major peak was
isolated.
N-(9-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((2S,-
3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2-yl)-2-sulfidohexahydrobenzo[d][1,3,2]-
oxathiaphosphol-2-yl)oxy)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin--
2-yl)isobutyramide (5.8 g, 6.55 mmol, 47.1% yield). .sup.1H NMR
(500 MHz, ACETONITRILE-d.sub.3) .delta. 12.06-11.78 (m, 1H), 9.22
(br s, 1H), 7.82 (s, 1H), 7.46-7.34 (m, 2H), 7.34-7.16 (m, 8H),
6.90-6.72 (m, 4H), 6.26 (t, J=6.8 Hz, 1H), 5.54-5.42 (m, 1H), 4.99
(d, J=1.2 Hz, 1H), 4.87 (s, 1H), 4.43 (dt, J=12.7, 3.2 Hz, 1H),
4.30 (dt, J=5.8, 3.1 Hz, 1H), 4.09 (q, J=7.1 Hz, 1H), 3.76 (d,
J=2.9 Hz, 6H), 3.45 (dd, J=10.5, 5.9 Hz, 1H), 3.29 (dd, J=10.4, 3.5
Hz, 1H), 3.15-3.02 (m, 1H), 2.70-2.53 (m, 3H), 2.20 (s, 8H),
2.03-1.91 (m, 7H), 1.76 (s, 3H), 1.67 (s, 3H), 1.17 (t, J=7.2 Hz,
6H).
2.20. Compound 3-131
##STR00462##
[0712] A dry 250 mL round bottom flask was charged with
(2R,3aR,6S,7aR)-3a-methyl-2-((perfluorophenyl)thio)-6-(prop-1-en-2-yl)hex-
ahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide (5.33 g, 11.94
mmol) and
1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)phenyl)methoxy)methyl)-4-hydroxytet-
rahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (5 g, 9.18
mmol) under a stream of nitrogen. N,N-Dimethylformamide (40 mL) was
added to the mixture which was allowed to stir at room temperature
until it became homogeneous. The mixture was cooled to 0.degree. C.
1,8-diazabicyclo[5.4.0]undec-7-ene (1.785 mL, 11.94 mmol) was then
added to the mixture drop-wise. After 10 minutes, the cold reaction
mixture was diluted with .about.200 mL toluene. The mixture was
transferred to a separatory funnel where it was washed twice with
1M pH 7.4 phosphate buffer solution (Aldrich P3619). Organics were
dried Na.sub.2SO.sub.4. The mixture was filtered and the filtrate
was concentrated to dryness. 9.25 g. The filtrate was evaporated
onto celite (toluene) The mixture was purified via Biotage medium
pressure liquid chromatography (Silica; Isco RediSep 24 g; 40%
EtOAc-Hex 1% triethylamine to 100% Ethyl acetate 1% triethylamine
over 16 CV). (4.3 g,
1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((2S,3aR-
,6S,7aR)-3a-methyl-6-(prop-1-en-2-yl)-2-sulfidohexahydrobenzo[d][1,3,2]oxa-
thiaphosphol-2-yl)oxy)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)--
dione (4.3 g, 59% yield). .sup.1H NMR (500 MHz,
ACETONITRILE-d.sub.3) .delta. 9.25-9.00 (m, 1H), 7.55-7.42 (m, 3H),
7.39-7.30 (m, 6H), 7.30-7.23 (m, 1H), 7.01-6.78 (m, 4H), 6.27 (dd,
1=8.1, 6.1 Hz, 1H), 5.55-5.42 (m, 1H), 4.98 (d, J=1.1 Hz, 1H), 4.87
(s, 1H), 4.44 (dt, J=12.7, 3.3 Hz, 1H), 4.21 (q, J=3.0 Hz, 1H),
3.79 (s, 7H), 3.51-3.28 (m, 2H), 2.62 (br s, 1H), 2.53-2.38 (m,
2H), 2.31-2.11 (m, 8H), 2.11-2.02 (m, 1H), 2.02-1.75 (m, 11H),
1.75-1.61 (m, 4H), 1.55-1.45 (m, 3H). .sup.31P NMR (202 MHz,
ACETONITRILE-d.sub.3) .delta. 100.4 (br d, J=10.0 Hz, 1P).
Example 17
1. Synthesis of 3'-O-Protected Nucleosides (General Procedure
1)
##STR00463##
[0714] 3'-OTBDPS protected nucleosides were prepared from the
commercially available 5'-ODMTr nucleosides according to the
following procedures.
1.1 Intermediate 17-i
##STR00464##
[0716] To a solution of 5'-O-(4,4'-dimethoxytrityl)thymidine (15.0
g, 27.6 mmol, 1.0 equiv.) and imidazole (3.14 g, 46.2 mmol, 2.0
equiv.) in DMF (30 mL) was added TBDPS-Cl (7.39 g, 33.1 mmol, 1.2
equiv.) at ambient temperature and stirred for 3 days. The
resulting mixture was then poured into water (0.7 L), and mixed for
0.5 h. The slurry was filtered and the cake was washed with water,
and then hexanes. The filter cake was dissolved in DCM. The
resulting solution washed with 5% aqueous citric acid solution and
dried over MgSO.sub.4. To the resulting DCM stream was added
dichloroacetic acid (11.4 mL) and Et.sub.3SiH (18 mL). After
stirring for 18 h, the mixture was quench with saturated aqueous
NaHCO.sub.3 and heptane. The isolated organic layer was
concentrated, and the resulting residue was purified by
chromatography (EtOAc/DCM). Intermediate 17-1 (10.1 g, 75%) was
isolated as a white solid with spectral characteristics consistent
with the literature Gao. R., et al., Biochemistry 43, 6167-6181
(2004).
1.2 Intermediate 17-ii
##STR00465##
[0718] Intermediate 17-ii was prepared analogously to Intermediate
17-i using N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine
(15.0 g, 23.7 mmol, 1.0 equiv.). SI-7 (10.3 g, 73%) was isolated as
a white solid with spectral characteristics consistent with the
literature Gao, R., et al., Biochemistry 43, 6167-6181 (2004).
1.3 Intermediate 17-iii
##STR00466##
[0719] Intermediate 17-iii was prepared analogously to Intermediate
17-1 using
N.sup.2-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine
(15.0 g, 23.5 mmol, 1.0 equiv.). Intermediate 17-iii (9.8 g, 75%)
was isolated as a white solid with spectral characteristics
consistent with the literature (Gao, R., et al., Biochemistry 43,
6167-6181 (2004)).
1.4 Intermediate 17-iv
##STR00467##
[0721] Intermediate 17-iv was prepared analogously to Intermediate
17-1 using N6-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine
(35.0 g, 53.3 mmol, 1.0 equiv.). Intermediate 17-iv (17.8 g, 56%)
was isolated as a white solid with spectral characteristics
consistent with the literature (Gao, R., et al., Biochemistry 43,
6167-6181 (2004)).
2. Synthesis of Dinucleotides
2.1 Compound 5-1
##STR00468##
[0723] To a 50 mL flask were added compound 3-107 (535 mg, 0.73
mmol, 1.0 equiv.) and nucleoside
N-(9-((2R,4S,5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(hydroxymethyl)tetrah-
ydrofuran-2-yl)-9H-purin-6-yl)benzamide (intermediate 17-iv) (0.88
g, 1.5 mmol, 2.0 equiv.) in THF (5 mL). DBU (0.33 mL, 2.19 mmol, 3
equiv.) was then added dropwise and the reaction was left to stir
at ambient temperature. After 10 minutes UPLC analysis showed
complete consumption of compound 3-107. The reaction mixture was
diluted with EtOAc (10 mL), DCM (5 mL) and 20% citric acid (5 mL).
The organic phase was washed with brine (5 mL), dried over
Na.sub.2SO.sub.4, filtered and the solvent was removed in vacuo.
The crude residue was purified by silica gel column chromatography
(0 to 100% MeOH in DCM) to afford the compound 5-1 as a white solid
(548 mg, 65%).
[0724] Physical State: White solid;
[0725] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 11.1 (br s,
1H), 8.84 (s, 1H), 8.71 (s, 1H), 8.21 (s, 1H), 8.08 (s, 1H),
8.05-8.00 (m, 2H), 7.72-7.47 (m, 12H), 7.45-7.27 (m, 11H), 6.62
(dd, J=8.5, 5.9 Hz, 1H), 6.27 (dd, J=8.5, 6.0 Hz, 1H), 5.10 (br dd,
J=6.2, 3.4 Hz, 1H), 4.69 (br s, 1H), 4.19 (br d, J=16.4 Hz, 2H),
3.89 (br dd, J=11.1, 4.0 Hz, 2H), 3.80-3.66 (m, 2H), 2.82-2.65 (m,
2H), 2.48-2.23 (m, 2H), 1.06 (s, 9H), 0.93 (s, 9H);
[0726] .sup.13C NMR (101 MHz, DMSO-d.sub.6): .delta. 151.9, 150.1,
148.8, 142.9, 139.1, 135.1, 134.9, 134.8, 134.3, 133.3, 132.7,
132.6, 132.6, 132.4, 132.2, 129.9, 129.8, 129.6, 129.6, 128.3,
128.2, 127.7, 127.6, 127.4, 125.3, 118.9, 86.4, 85.9, 83.4, 75.1,
74.6, 64.8, 64.7, 63.9, 48.4, 40.2, 39.9, 37.3, 30.5, 26.6, 26.5,
26.4, 18.6, 18.5;
[0727] .sup.31P NMR (162 MHz, DMSO-d.sub.6): .delta. 53.8;
[0728] HRMS (ESI-TOF, m/z): Calcd for
[C.sub.59H.sub.65H.sub.10O.sub.8PSSi.sub.2+H].sup.+ 1161.4057;
Found 1161.4094 (3.3 ppm error).
[0729] R.sub.f=0.30 (10% MeOH in DCM); UV, KMnO.sub.4.
2.2. Compound 5-2
##STR00469##
[0731] To a 50 mL flask were added compound 3-108 (1.00 g, 1.36
mmol, 1.0 equiv.) and nucleoside
N-(1-((2R,4S,5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(hydroxymethyl)tetrah-
ydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide
(intermediate 17-ii) (1.55 g, 2.72 mmol, 2.0 equiv.) in THF (3 mL)
and MeCN (20 mL). DBU (0.41 mL, 2.72 mmol, 2 equiv.) was then added
dropwise and the reaction was left to stir at ambient temperature.
After 1 hour UPLC analysis showed complete consumption of compound
3-108. The reaction mixture was diluted with EtOAc (25 mL) and 20%
citric acid (25 mL). Then the organic phase was washed with brine
(25 mL), dried over Na.sub.2SO.sub.4, filtered and the solvent was
removed in vacuo. The crude residue was purified by silica gel
column chromatography (0 to 35% MeOH in EtOAc) to afford the
product 5-2 as a white solid (1.41 g, 91%).
[0732] Physical State: White solid;
[0733] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 11.21 (br s,
1H), 8.55 (d, J=7.3 Hz, 1H), 8.22 (s, 1H), 8.09 (s, 1H), 8.00-7.95
(m, 2H), 7.63-7.54 (m, 9H), 7.53-7.26 (m, 17H), 6.38 (t, J=6.5 Hz,
1H), 6.32 (t, J=6.7 Hz, 1H), 5.18-5.11 (m, 1H), 4.48 (br d, J=4.3
Hz, 1H), 4.23-4.03 (m, 2H), 3.91-3.78 (m, 2H), 3.73 (br dd, J=11.1,
4.8 Hz, 1H), 3.68-3.52 (m, 1H), 2.89-2.78 (m, 1H), 2.57-2.50 (m,
1H), 2.28 (hr dd, J=12.5, 5.4 Hz, 1H), 1.94 (ddd, J=13.3, 8.3, 5.2
Hz, 1H), 1.02 (s, 9H), 0.93 (s, 9H); .sup.13C NMR (101 MHz,
DMSO-d.sub.6): .delta. 167.3, 163.0, 155.3, 154.4, 151.7, 149.1,
145.5, 139.1, 136.4, 135.2, 135.2, 135.1, 135.0, 134.5, 133.2,
132.8, 132.7, 132.7, 132.5, 130.0, 129.8, 129.7, 129.2, 128.4,
128.0, 128.0, 127.8, 127.7, 127.5, 119.1, 96.7, 86.8, 86.8, 86.3,
85.9, 85.8, 83.6, 75.0, 75.0, 74.5, 64.5, 64.4, 64.1, 41.2, 38.0,
26.7, 26.6, 18.7, 18.6;
[0734] .sup.31P NMR (162 MHz, DMSO-d.sub.6): .delta. 54.1;
[0735] HRMS (ESI-TOF, m/z): Calcd for
[C.sub.58H.sub.65N.sub.8O.sub.9PSSi.sub.2+H].sup.+ 1137.3944; Found
1137.3969 (2.1 ppm error).
2.3. Compound 5-3
##STR00470##
[0737] To a 50 mL flask were added compound 3-108 (1.01 g, 1.37
mmol, 1.0 equiv.) and nucleoside
1-((2R,4S,5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(hydroxymethyl)tetrahydr-
ofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (Intermediate
17-i) (1.15 g, 2.39 mmol, 1.8 equiv.) in THF (10 mL). DBU (0.55 mL,
3.70 mmol, 2.7 equiv.) was then added dropwise and the reaction was
left to stir at ambient temperature. After 10 minutes UPLC analysis
showed complete consumption of compound 3-108. The reaction mixture
was diluted with EtOAc (10 mL), DCM (5 mL), and 20% citric acid (5
mL). The organic phase was washed with brine (5 mL), dried over
Na.sub.2SO.sub.4, filtered, and the solvent was removed in vacuo.
The crude residue was purified by silica gel column chromatography
(0 to 100% MeOH in DCM) to afford the compound 5-3 as a white solid
(0.88 g, 61%).
[0738] Physical State: White solid;
[0739] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 11.24 (s, 1H),
8.29 (s, 1H), 8.14 (s, 1H), 7.67-7.58 (m, 1H), 7.58-7.48 (m, 9H),
7.43-7.23 (m, 12H), 6.36-6.22 (m, 2H), 5.12 (br d, J=2.8 Hz, 1H),
4.48-4.33 (m, 1H), 4.09-3.90 (m, 2H), 3.81 (hr dd, J=11.1, 4.3 Hz,
1H), 3.76-3.51 (m, 3H), 2.79-2.83 (m, 1H), 2.71 (d, J=15.4 Hz, 1H),
2.61 (d, J=15.4 Hz, 1H), 2.03-1.99 (m, 1H), 1.72 (s, 3H), 0.97 (s,
9H), 0.89 (s, 9H);
[0740] .sup.13C NMR (101 MHz, DMSO-d.sub.6): .delta. 174.5, 171.2,
163.6, 150.5, 148.5, 135.8, 135.2, 135.1, 135.0, 135.0, 134.4,
132.7, 132.6, 132.4, 130.0, 129.8, 129.8, 128.0, 127.9, 127.8,
127.7, 127.5, 119.0, 110.0, 85.7, 83.9, 83.7, 74.3, 72.4, 48.6,
42.6, 40.9, 40.8, 40.7, 40.4, 26.7, 26.6, 18.7, 18.6, 14.1,
12.1;
[0741] .sup.31P NMR (162 MHz, DMSO-d.sub.6): .delta. 57.0;
[0742] HRMS (ESI-TOF, m/z): Calcd for
[C.sub.52H.sub.62N.sub.7O.sub.9PSSi.sub.2+H].sup.+ 1048.3679; Found
1048.3705 (2.5 ppm error).
2.4. Compound 5-4
##STR00471##
[0744] To a 50 mL flask were added compound 3-108 (803 mg, 1.09
mmol, 1.0 equiv.) and nucleoside
N-(9-((2R,4S,5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(hydroxymethyl)tetrah-
ydrofuran-2-yl)-6-hydroxy-9H-purin-2-yl)isobutyramide (Intermediate
17-iii) (1.24 g, 2.18 mmol, 2.0 equiv.) in THF (10 mL). DBU (0.47
mL, 3.27 mmol, 3 equiv.) was then added dropwise and the reaction
was left to stir at ambient temperature. After 30 minutes UPLC
analysis showed complete consumption of compound 3-108. The
reaction mixture was diluted with EtOAc (20 mL) and 20% citric acid
(10 mL). Then the organic phase was washed with brine (5 mL), dried
over Na.sub.2SO.sub.4, filtered, and the solvent was removed in
vacuo. The crude residue was purified by silica gel column
chromatography (0 to 20% MeOH in DCM) to afford the compound 5-4 as
a white solid (986 mg, 79%).
[0745] Physical State: White solid;
[0746] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 12.22 (s, 1H),
12.08 (s, 1H), 8.30 (s, 1H), 8.20 (s, 1H), 8.08 (s, 1H), 7.80-7.21
(br m, 23H), 6.37 (dd, J=9.4, 5.3 Hz, 1H), 6.27 (dd, J=8.1, 6.1 Hz,
1H), 5.21-5.09 (br m, 1H), 4.75 (hr d, J=4.6 Hz, 1H), 4.19-4.05
(br, 2H), 3.95-3.69 (hr m, 4H), 3.08-2.93 (hr m, 1H), 2.92-2.77 (m,
2H), 2.17 (hr dd, J=12.6, 5.3 Hz, 1H), 1.10 (d, J=6.8 Hz, 3H), 1.07
(s, 9H), 1.05 (d, J=6.8 Hz, 3H), 0.95 (s, 9H);
[0747] .sup.13C NMR (101 MHz, DMSO-d.sub.6): .delta. 180.3, 155.6,
154.9, 152.0, 149.1, 148.5, 147.6, 139.1, 139.0, 135.3, 135.2,
135.1, 135.0, 133.0, 132.8, 132.7, 132.5, 130.0, 129.8, 129.7,
128.0, 127.9, 127.8, 127.7, 120.8, 119.1, 87.0, 86.9, 85.7, 85.6,
84.7, 83.5, 83.4, 75.0, 74.7, 74.6, 64.6, 64.5, 64.0, 37.7, 34.5,
26.8, 26.6, 18.9, 18.7, 18.7, 18.6;
[0748] .sup.31P NMR (162 MHz, DMSO-d.sub.6): .delta. 54.3;
[0749] HRMS (ESI-TOF, m/z): Calcd for
[C.sub.56H.sub.67N.sub.10O.sub.9PSSi.sub.2+H].sup.+ 1143.4162,
Found 1143.4186 (2.1 ppm error).
2.5. Compound 5-5
##STR00472##
[0751] To a 50 mL flask were added compound 3-109 (1.02 g, 1.43
mmol, 1.0 equiv.) and nucleoside
N-(1-((2R,4S,5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(hydroxymethyl)tetrah-
ydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide
(Intermediate ii) (1.60 g, 2.86 mmol, 2.0 equiv.) in MeCN (20 mL)
and THF (5 mL). DBU (0.32 mL, 2.15 mmol, 3 equiv.) was then added
dropwise and the reaction was left to stir at ambient temperature.
After 1 hour UPLC analysis showed complete consumption of compound
3-109. The reaction mixture was diluted with EtOAc (25 mL) and 20%
citric acid (20 mL). Then the organic phase was washed with brine
(10 mL), dried over Na.sub.2SO.sub.4, filtered, and the solvent was
removed in vacuo. The crude residue was purified by silica gel
column chromatography (20% MeOH in DCM) to afford the product 5-5
as a white solid (1.23 g, 73%).
[0752] Physical State: White solid;
[0753] .sup.1H NMR (500 MHz, DMSO-d.sub.6): .delta. 11.20 (br s,
1H), 9.23 (br s, 1H), 8.47 (br d, J=7.5 Hz, 1H), 8.21 (br s, 1H),
7.98 (d, J=7.8 Hz, 2H), 7.90 (d, J=7.8 Hz, 1H), 7.64-7.58 (m, 9H),
7.52-7.37 (m, 14H), 7.29 (br s, 1H), 6.33 (t, J=7.0 Hz, 1H), 6.08
(t, J=6.5 Hz, 1H), 5.81 (d, J=7.8 Hz, 1H), 4.96 (hr d, J=2.9 Hz,
1H), 4.52-4.45 (m, 1H), 4.16 (hr s, 2H), 3.92-3.85 (m, 2H),
3.85-3.71 (m, 1H), 3.70-3.25 (m, 2H), 2.48-2.28 (m, 2H), 2.14 (dt,
J=13.7, 6.7 Hz, 1H), 2.00-1.90 (m, 1H), 1.05 (s, 9H), 0.96 (s,
9H);
[0754] .sup.13C NMR (126 MHz, DMSO-d.sub.6): .delta. 162.9, 159.8,
148.0, 145.2, 143.0, 135.2, 135.2, 135.1, 134.9, 134.4, 133.1,
132.7, 132.7, 132.6, 132.1, 129.9, 129.1, 128.1, 127.9, 127.5,
96.4, 93.9, 86.7, 86.1, 86.3, 85.7, 74.3, 74.2, 64.5, 63.7, 41.1,
26.7, 26.6, 18.7, 18.6;
[0755] .sup.31P NMR (202 MHz, DMSO-d.sub.6): .delta. 54.6;
[0756] HRMS (ESI-TOF, m/z): Calcd for
[C.sub.57HN.sub.65O.sub.10PSSi.sub.2+H].sup.+ 1113.3832; Found
1113.3859 (2.4 ppm error).
2.6. Compound 5-6
##STR00473##
[0758] To a 50 mL flask were added compound 3-110 (1.00 g, 1.40
mmol, 1.0 equiv.) and nucleoside
1-((2R,4S,5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(hydroxymethyl)tetrahydr-
ofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (Intermediate
17-i) (1.35 g, 2.80 mmol, 2.0 equiv.) in MeCN (20 mL) and THF (5
mL). DBU (0.33 mL, 2.10 mmol, 3 equiv.) was then added dropwise and
the reaction was left to stir at ambient temperature. After 1 hour
UPLC analysis showed complete consumption of compound 3-110. The
reaction mixture was diluted with EtOAc (25 mL) and 20% citric acid
(20 mL). Then the organic phase was washed with brine (10 mL),
dried over Na.sub.2SO.sub.4, filtered and the solvent was removed
in vacuo. The crude residue was purified by silica gel column
chromatography (20% MeOH in DCM) to afford Compound 5-6 as a white
solid (1.12 g, 76%).
[0759] Physical State: White solid;
[0760] .sup.1H NMR (500 MHz, DMSO-d.sub.6): .delta. 11.25 (s, 1H),
8.36 (br s, 1H), 7.84-7.71 (m, 3H), 7.63 (br t, J=6.3 Hz, 4H), 7.57
(hr s, 4H), 7.48-7.36 (m, 12H), 6.36 (hr t, J=7.2 Hz, 1H), 6.08 (br
t, J=6.4 Hz, 1H), 5.71 (hr d, J=7.3 Hz, 1H), 4.98 (hr s, 1H), 4.45
(br s, 1H), 3.95 (br d, J=17.9 Hz, 2H), 3.81-3.76 (m, 1H),
3.73-3.65 (m, 2H), 3.57-3.50 (m, 2H), 2.40-2.31 (m, 1H), 2.12-2.04
(m, 1H), 2.04-1.92 (m, 2H), 1.81 (s, 3H), 1.02 (s, 9H), 0.97 (s,
9H);
[0761] .sup.13C NMR (126 MHz, DMSO-d.sub.6): .delta. 164.2, 162.9,
151.6, 151.0, 142.2, 136.5, 135.7, 135.6, 135.4, 133.2, 133.1,
132.6, 130.5, 130.4, 128.4, 110.6, 94.3, 86.5, 86.0, 85.7, 84.3,
75.3, 74.4, 72.9, 65.0, 64.2, 27.2, 27.1, 19.2, 19.0, 12.5;
[0762] .sup.31P NMR (202 MHz, DMSO-d.sub.6): .delta. 53.5;
[0763] HRMS (ESI-TOF, m/z): Calcd for
[C.sub.51H.sub.62N.sub.5O.sub.10PSSi.sub.2+H].sup.+ 1024.3566,
Found 1024.3600 (3.3 ppm error).
2.7. Compound 5-7
##STR00474##
[0765] A mixture of compound 3-109 (1.00 g, 1.35 mmol, 1.00 equiv.)
and the nucleoside
N-(9-((2R,4S,5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(hydroxymethyl)tetrah-
ydrofuran-2-yl)-6-hydroxy-9H-purin-2-yl)isobutyramide (Intermediate
17-iii) (1.55 g, 2.70 mmol, 2.0 equiv.) was dissolved in a mixture
of THF (10 mL) and MeCN (20 mL), then concentrated in vacuo
(3.times.). The residue was dissolved in a mixture of THF (10 mL)
and MeCN (20 mL) and DBU (608 .mu.L, 4.04 mmol, 3.0 equiv.) was
added. The reaction mixture was stirred for 30 min, then diluted
with EtOAc (20 mL) and aqueous 1 N HCl (20 mL, pH=1). The layers
were separated and the aqueous layer was extracted with EtOAc
(2.times.20 mL). The combined organic extracts were dried over
sodium sulfate, filtered, and concentrated in vacuo to afford a
gel. The gel was purified by flash column chromatography (5 to 20%
MeOH in DCM, performed twice). The desired compound 5-7 was
isolated as a gel, which was stirred in MTBE (40 mL) for 1 h to
convert the product to a white powder which was isolated by
filtration (1.23 g, 82%).
[0766] Physical State: White solid;
[0767] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 12.31 (br s,
1H), 12.08 (br s, 1H), 9.23 (br s, 1H), 8.25 (br s, 2H), 7.90 (d,
J=7.8 Hz, 1H), 7.53-7.70 (m, 9H), 7.35-7.49 (m, 13H), 6.35 (dd,
J=9.5, 5.4 Hz, 1H), 6.04 (t, J=6.5 Hz, 1H), 5.80 (d, J=7.8 Hz, 1H),
4.94 (dt, J=6.0, 2.9 Hz, 1H), 4.57 (br d, J=4.5 Hz, 1H), 4.08-4.11
(m, 2H), 3.75-3.93 (m, 3H), 3.53-3.58 (m, 3H), 2.76-2.96 (m, 2H),
2.24-2.33 (m, 1H), 2.26-2.20 (m, 2H), 1.05-1.10 (m, 16H), 0.97 (s,
9H);
[0768] .sup.13C NMR (101 MHz, DMSO-d.sub.6): .delta. 180.4, 159.7,
154.9, 148.5, 147.86, 147.82, 143.1, 138.5, 135.25, 135.20, 135.1,
134.9, 132.74, 132.70, 132.6, 129.98, 129.94, 129.90, 127.94,
127.88, 120.6, 94.0, 86.60, 86.53, 86.13, 85.8, 84.3, 74.7, 74.2,
64.8, 63.7, 34.6, 26.7, 26.6, 18.8, 18.7, 18.6;
[0769] .sup.31P NMR (162 MHz, DMSO-d): .delta. 54.78;
[0770] HRMS (ESI-TOF, m/z): Calcd for
[C.sub.55H.sub.67N.sub.8O.sub.10PSSi.sub.2+H].sup.+1119.4050; Found
1119.4071 (1.9 ppm error).
2.8. Compound 5-8
##STR00475##
[0772] To a solution of the compound 3-112 (1.00 g, 1.26 mmol, 1.0
equiv.) and nucleoside
1-((2R,4S,5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(hydroxymethyl)tetrahydr-
ofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (Intermediate
17-i) (1.17 g, 1.17 g, 2.52 mmol, 2.0 equiv.) in THF (10 mL) was
added DBU (581 .mu.L, 3.78 mmol, 3.0 equiv.). The reaction mixture
was stirred for 30 min, then diluted with EtOAc (10 mL) and aqueous
1 N HCl (5 mL, pH=1). The layers were separated and the aqueous
layer was extracted with EtOAc (10 mL). The combined organic
extracts were dried over Na.sub.2SO.sub.4, filtered, then
concentrated in vacuo to afford a gel. The gel was purified by
flash column chromatography (0 to 10% to 30% MeOH in DCM to 30%,
performed twice). The desired compound 5-8 was isolated as a gel,
which was concentrated from hexanes (10 mL) to afford a white
powder (926.1 mg, 72%).
[0773] Physical State: White solid;
[0774] .sup.1H NMR (400 MHz, DMSO-d): .delta. 11.37 (s, 1H), 11.26
(s, 1H), 7.84 (s, 1H), 7.61-7.66 (m, 5H), 7.55-7.57 (m, 4H),
7.35-7.45 (m, 13H), 6.35 (dd, J=8.5, 6.2 Hz, 1H), 6.13 (dd, J=8.8,
5.6 Hz, 1H), 5.03-5.07 (m, 1H), 4.40 (m, 1H), 3.94 (brd, J=9.3 Hz,
2H), 3.65-3.76 (m, 3H), 3.41-3.53 (m, 1H), 2.24 (br dd, J=12.9, 5.1
Hz, 1H), 2.03-2.14 (m, 1H), 1.90-2.03 (m, 2H), 1.82 (s, 3H), 1.43
(s, 3H), 1.01 (s, 9H), 0.98 (s, 9H);
[0775] .sup.13C NMR (101 MHz. DMSO-d.sub.6): .delta. 163.8, 163.6,
150.6, 150.4, 136.1, 135.20, 135.18, 135.1, 135.0, 134.9, 132.8,
132.72, 132.68, 132.0, 130.03, 129.96, 127.96, 127.91, 110.2,
109.7, 86.2, 86.1, 85.4, 85.3, 83.9, 74.97, 74.4, 64.2, 38.7, 26.7,
26.6, 18.8, 18.5, 12.1, 11.8;
[0776] .sup.31P NMR (162 MHz, DMSO-d.sub.6): .delta. 53.01;
[0777] HRMS (ESI-TOF, m/z): Calcd for
[C.sub.52H.sub.63N.sub.4O.sub.11PSSi.sub.2+H].sup.+ 1039.3563;
Found 1039.3586 (2.3 ppm error).
2.9. Compound 5-9
##STR00476##
[0779] To a mixture of compound 3-112 (734 mg, 1.0 equiv.) and
N-(9-((2R,4S,5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(hydroxymethyl)tetrah-
ydrofuran-2-yl)-6-hydroxy-9H-purin-2-yl)isobutyramide (Intermediate
17-iii) (1.20 g, 2.1 equiv.) in THF was added DBU (0.45 mL, 3.0
equiv.). After 30 min, EtOAc (20 mL) and 20% citric acid (10 mL)
were added. The phases were separated and the organic phase was
washed with brine (5 mL) and dried over Na.sub.2SO.sub.4. The
solution was filtered and the solvent was removed under vacuum. The
residue was dissolved with DCM, and purified by ISCO flash
chromatography (80 g silica gel column, 0 to 20% MeOH in DCM, 18
min run). The pure fractions were combined and solvents were
removed to afford the desired compound 5-9 (380 mg) as white solid.
Then, the impure fractions were combined, solvents were removed in
vacuo and a second ISCO flash chromatography was performed using
the same conditions. The desired compound 5-9(388 mg) was obtained
as white solid. In total, compound 5-9(768 mg, 67%) was
obtained.
[0780] Physical State: White solid;
[0781] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 12.17 (s, 1H),
12.06 (s, 1H), 11.37 (s, 1H), 8.30 (s, 1H), 7.77-7.35 (br m, 21H),
6.35 (dd, J=9.6, 5.3 Hz, 1H), 6.13 (dd, J=8.8, 5.6 Hz, 1H),
5.16-5.01 (br m, 1H), 4.71 (br d, J=4.6 Hz, 1H), 4.12-4.04 (br m,
1H), 4.02-3.93 (br m, 1H), 3.86-3.73 (br m, 3H), 3.72-3.63 (br m,
1H), 3.02-2.89 (br m, 1H), 2.87-2.76 (br m, 1H), 2.30 (br dd,
J=13.1, 6.8 Hz, 1H), 2.20-2.04 (br m, 2H), 1.44 (s, 3H), 1.12 (d,
J=6.8 Hz, 3H), 1.10 (d, J=6.8 Hz, 3H), 1.06 (s, 9H), 0.99 (s,
9H);
[0782] .sup.13C NMR (101 MHz, DMSO-d.sub.6): .delta. 180.3, 163.6,
154.9, 150.3, 148.5, 147.6, 139.1, 135.3, 135.2, 135.1, 135.0,
134.8, 132.9, 132.8, 132.8, 132.1, 129.9, 128.0, 127.9, 120.7,
109.6, 87.0, 86.9, 85.3, 85.2, 84.6, 83.8, 75.0, 74.7, 74.6, 64.3,
64.2, 64.1, 54.9, 38.7, 38.4, 34.5, 26.8, 26.6, 18.9, 18.8, 18.7,
18.6, 11.7;
[0783] .sup.31P NMR (162 MHz, DMSO-d.sub.6): .delta. 54.1;
[0784] HRMS (ESI-TOF, m/z): Calcd for [C56H68N7O11PSSi2+H]+
1134.4046, Found 1134.4080 (2.9 ppm error).
2.10. Compound 5-10
##STR00477##
[0786] To a 50 mL flask were added compound 3-113 (1.00 g, 1.24
mmol, 1.0 equiv.) and nucleoside
N-(9-((2R,4S,5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(hydroxymethyl)tetrah-
ydrofuran-2-yl)-6-hydroxy-9H-purin-2-yl)isobutyramide (Intermediate
17-iii) (1.42 g, 2.48 mmol, 2.0 equiv.) in MeCN (20 mL) and THF (3
mL). DBU (0.37 mL, 2.48 mmol, 2 equiv.) was then added dropwise and
the reaction was left to stir at ambient temperature. After 2 hour
UPLC analysis showed complete consumption of compound 3-113. The
reaction mixture was diluted with EtOAc (25 mL) and 20% citric acid
(25 mL) and was allowed to stir at ambient temperature for 4 hours.
The dimethylaminomethylene was converted to formamide based on
HPLC-MS analysis. Then the organic phase was washed with brine (25
mL), dried over Na.sub.2SO.sub.4, filtered and the solvent was
removed in vacuo. The crude residue was purified by silica gel
column chromatography (0 to 60% MeOH in EtOAc) to afford the
compound 5-10 as a white solid (1.27 g, 86%).
[0787] Physical State: White solid;
[0788] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 12.33 (br s,
1H), 12.10 (s, 1H), 8.28 (s, 1H), 8.00 (s, 1H), 7.78-7.63 (m, 1H),
7.63-7.54 (m, 8H), 7.46-7.25 (m, 13H), 6.36 (dd, J=9.2, 5.4 Hz,
1H), 6.14 (t, J=6.6 Hz, 1H), 5.05 (br s, 1H), 4.64-4.55 (m, 1H),
4.14 (br s, 2H), 4.00-3.88 (m, 1H), 3.88-3.70 (m, 2H), 3.67-3.55
(m, 1H), 2.96-2.74 (m, 2H), 2.58-2.50 (m, 1H), 2.33-2.14 (m, 2H),
1.12-1.05 (m, 6H), 1.02 (s, 9H), 0.93 (s, 9H);
[0789] .sup.13C NMR (101 MHz, DMSO-d.sub.6): .delta. .delta. 180.5,
154.9, 148.5, 148.3, 147.8, 147.2, 139.1, 137.3, 135.2, 135.2,
135.1, 135.0, 132.8, 132.5, 130.0, 129.8, 129.7, 128.0, 127.9,
127.8, 127.7, 120.8, 120.6, 86.7, 86.6, 86.2, 86.2, 84.8, 83.3,
74.8, 74.6, 64.7, 64.3, 48.6, 37.9, 34.6, 26.8, 26.6, 18.9, 18.8,
18.6;
[0790] .sup.31P NMR (162 MHz, DMSO-d.sub.6): .delta. 54.2; 1
[0791] HRMS (ESI-TOF, m/z): Calcd for
[CH.sub.57H.sub.67N.sub.10O.sub.11PSSi.sub.2+H].sup.+ 1187.4060;
Found 1187.4085 (2.0 ppm error).
[0792] The table below lists UPLC/HPLC conditions and retention
times for compounds above.
TABLE-US-00011 UPLC/HPLC conditions and retention times Retention
Entry Product Assay Conditions time 1 ##STR00478## UPLC Ascentis
express C18 2.7 um 2.1 x 50 mm Solvent A: 0.05% TFA in
MeCN:H.sub.2O (5:95) Solvent B: 0.05% TFA in MeCN:H.sub.2O (95:5)
Gradient: Complex--0% to 100% B over 2 min Flow rate: 1 mL/min PDA
wavelength: 220 nm 1.91 2 ##STR00479## UPLC Ascentis express C18
2.6 um 2.1 x 50 mm Solvent A: 0.05% TFA in MeCN:H.sub.2O (5:95)
Solvent B: 0.05% TFA in MeCN:H.sub.2O (95:5) Gradient: Complex--0%
to 100% B over 2 min Flow rate: 1 mL/min PDA wavelength: 220 nm
2.08 3 ##STR00480## UPLC Ascentis express C18 2.7 um 2.1 x 50 mm
Solvent A: 0.05% TFA in MeCN:H.sub.2O (5:95) Solvent B: 0.05% TFA
in MeCN:H.sub.2O (95:5) Gradient: Complex--10% to 100% B over 2 min
Flow rate: 1 mL/min PDA wavelength: 220 nm 1.88 4 ##STR00481## UPLC
Ascentis express C18 2.7 um 2.1 x 50 mm Solvent A: 0.01%
NH.sub.4OAc in MeCN:H.sub.2O (5:95) Solvent B: 0.01% NH.sub.4OAc in
MeCN:H.sub.2O (95:5) Gradient: Complex--0% to 100% B over 2 min
Flow rate: 1 mL/min PDA wavelength: 220 nm 1.92 5 ##STR00482## UPLC
Thermo Accucore aQ 2.6 UM 2.1 x 50 MITI Solvent A: 0.01%
NH.sub.4OAc in MeCN:H.sub.2O (5:95) Solvent B: 0.01% NH.sub.4OAc in
MeCN:H.sub.2O (95:5) Gradient: Complex--10% to 100% B over 2 min
Flow rate: 1 mL/min PDA wavelength: 220 nm 1.49 6 ##STR00483## UPLC
Ascentis express C18 2.7 um 2.1 x 50 mm Solvent A: 0.01%
NH.sub.4OAc in MeCN:H.sub.2O (5:95) Solvent B: 0.01% NH.sub.4OAc in
MeCN:H.sub.2O (95:5) Gradient: Complex--10% to 100% B over 2 min
Flow rate: 1 mL/min PDA wavelength: 220 nm 1.77 7 ##STR00484## HPLC
Xbridge BEH Shield RP18 2.5 um 4.6 x 50 mm Solvent A: 0.05% TFA in
MeOH:H.sub.2O (20:80) Solvent B: 0.05% TFA in MeOH:ACN (20:80)
Gradient: Complex--0% to 100% B over 30 min Flow rate: 0.8 mL/min
PDA wavelength: 220 nm, 256 nm 22.91 8 ##STR00485## HPLC Supelco
Ascentis express C18 2.7 um 4.6 x 150 mm Solvent A: 0.05% TFA in
MeOH:H.sub.2O (20:80) Solvent B: 0.05% TFA in MeOH:ACN (20:80)
Gradient: Complex--0% to 100% B over 30 min Flow rate: 1 mL/min PDA
wavelength: 220 nm, 256 nm 30.82 9 ##STR00486## UPLC Ascentis
express C18 2.7 um 2.1 x 50 mm Solvent A: 0.01% NH.sub.4OAc in
MeCN:H.sub.2O (5:95) Solvent B: 0.01% NH.sub.4OAc in MeCN:H.sub.2O
(95:5) Gradient: Complex--0% to 100% B over 2 min Flow rate: 1
mL/min PDA wavelength: 220 nm 2.04 10 ##STR00487## UPLC Agilent
Poroshell EC-C18 1.9 um 2.1 x 50 mm Solvent A: 0.01% NH4OAc in
MeCN:H.sub.2O (5:95) Solvent B: 0.01% NH.sub.4OAc in MeCN:H.sub.2O
(95:5) Gradient: Complex--0% to 100% B over 2 min Flow rate: 1
mL/min PDA wavelength: 220 nm 2.02
Example 18
Alternative Preparation of Phosphodiester Products
##STR00488##
##STR00489##
[0794] Variables of X, Y, and R are listed in the Table 8.
TABLE-US-00012 TABLE 8 X= OMe OMe OMe OMe H H Ph Ph Y= H F OMe Br F
Br H F R= H H H H H H H H # 1 2 3 4 5 6 7 8 X= Ph Ph H H H Cl H Br
H Y= OMe Br H Br H Cl OMe Br Cl R= H H Ph Ph H H H H H # 9 10 11 12
13 14 15 16 17
[0795] The reaction yields and times are listed in Table 9
TABLE-US-00013 TABLE 9 S-1 S-2 S-3 S-4 S-5 S-6 # Yield Yield Yield
Yield Yield Time 1 92% 67% 99% 74% 28% 0.5-1 hr 2 99%. 44% -- -- --
-- 3 31% 61% 96% -- -- -- 4 89% 67% 98% -- -- -- 5 63% 42% -- -- --
-- 6 99%. 85% -- -- -- -- 7 44% 84% -- -- -- -- 8 36% 45% -- -- --
-- 9 82% 95% -- -- -- -- 10 42% 88% -- -- -- -- 11 -- -- -- -- --
-- 12 -- -- -- -- -- -- 13 N/A 81% 79% 53% 60% 0.5-1 hr 14 N/A 76%
67% 54% 89% 0.5-1 hr 15 N/A 65% -- -- -- -- 16 96% 74% -- -- --
0.5-1 hr 17 N/A 89% 55% 62% 85% 0.2-0.5 hr
[0796] The detailed procedures below are for illustration, which
started from chalcone (18-iii) that is commercial available.
Step 2
##STR00490##
[0798] To a 1 L RBF was added chalcone (18-iii, 55 g, 0.26 mol, 1.0
equiv.) followed by thioacetic acid (55 mL, 1 v.). After 5 min, the
reaction became homogeneous. After stirring for 20 minutes, the
light yellow solution was diluted with methanol (550 mL, 10 v.).
The product immediately precipitated and this was allowed to stir
overnight. The solids were collected by filtration and dried. The
product was obtained as a white solid (61 g, 81% yield). 1H NMR
(400 MHz, Chloroform-d) .delta. 8.08-7.88 (m, 2H), 7.61-7.28 (m,
8H), 5.40-5.19 (m, 1H), 3.81-3.60 (m, 2H), 2.33 (t, J=3.0 Hz,
3H).
b. Step 3
##STR00491##
[0800] Lithium aluminum hydride (12.24 g, 1.5 equiv.) was slowly
dissolved in Et.sub.2O at 0.degree. C. To this was added a solution
of thioacetate (18-v, 61 g, 0.21 mol, 1.0 equiv.) in THF/Et.sub.2O
(200 mL, 1:1) dropwise over 1 hour at 0.degree. C. After the
addition was complete, solid LAH (1 g) was added. The reaction was
stirred overnight (HPLC analysis reavealed full conversion to
product). The reaction was quenced via the addition of ethyl
acetate dropwise (.about.100 mL) followed by saturated aqueous
potassium sodium tartrate and lastly 1 M NaOH. This was partitioned
between 1:1 ethyl acetate/hexane and additional water overnight.
The organic layer was decanted and additional solvent was added and
the mixing/decanting was repeated (2.times.1 L). The combined
organics were concentrated to .about.200 mL and dried over
MgSO.sub.4, filtered and concentrated. The product was obtained as
a colorless oil (18-vi, 41 g, 79% yield).
An Alternative Method to Make Intermediate 18-vi:
[0801] A RBF was charged with thiomichael adduct (5 g) followed by
THF (175 mL, 0.1 M) and cooled to 0.degree. C. LAH (1.3 g, 2.0
equiv.) was slowly added as a solid. After warming to rt overnight,
the reaction was quenched by slow addition of solid
Na.sub.2SO.sub.4 heptahydrate. After stirring for an hour the
reaction was filtered and concentrated to a clear oil. Note: this
was >80% pure by NMR. Quick flash chromatography provided the
mercaptopropanol as a colorless oil.
Another Alternative Method to Make Intermediate 18-vi:
[0802] To a solution of thioacetate (61 g, 0.21 mol, 1.0 equiv.) in
THF (200 mL) was added Lithium aluminum hydride (12.24 g, 1.5
equiv.) was slowly at 0.degree. C. The reaction was stirred
overnight (Reaction complete within 1 hour, HPLC analysis reavealed
full conversion to product). The reaction was quenced via the
addition of ethyl acetate dropwise (.about.100 mL) followed by
concentrated HCl or Sulfuric acid. This was partitioned between 1:1
ethyl acetate/hexane and water. The organic phase was washed with
additional HCl (1M) followed twice by water and lastly brine. The
combined organics were dried over MgSO4, filtered and concentrated.
The product was obtained as a colorless oil (41 g, 79% yield).
[0803] .sup.1H NMR (400 MHz, Chloroform-d) .delta. 7.49-7.20 (m,
10H), 4.96-4.48 (m, 1H), 4.21 (dtd, J=10.8, 8.7, 7.3, 3.8 Hz, 1H),
2.52-2.19 (m, 2H), 2.02 (ddd, J=15.1, 5.8, 1.8 Hz, 1H).
Procedures to Prepare an Enantiomer Enriched Intermediate
18-vi.
Procedure 1:
##STR00492##
[0805] 28 g 1,3-diphenyl-1,3-propanedinone was dissolved in 280 mL
of DCM. To this solution was added 23.6 mL of HCOOH, 34.5 mL of
TEA, and 3.89 g of Ru Catalyst. The reaction solution was refluxed
for 10 h. 280 mL of hexane was added, then the mixture was stirred
in ice bath for 10 min. The product was filtered and washed using
300 mL of DCM:Hexane (1:1). 19.8 g of product (18-xii or 18-xiii)
was obtained after dried in vacuum overnight. .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.7.40-7.27 (m, 10H), 5.01 (t, J=5.8 Hz, 2H),
2.25-2.18 (m, 2H).
Procedure 2:
##STR00493##
[0807] 20 mmol (4.56 g) of 1,3-diphenyl-dipropanediol (18-xii) and
20 mmol of Ts.sub.2O were dissolved in 100 mL of THF. Then the
temperature was decreased to -78.degree. C. After 20 mmol of 1M Li
tert-BuO/THF was added, the temperture was increased to room
temperature. After the temperature was decreased to -30.degree. C.,
22 mmol of TIPS (4.20 g) was added followed by another 20 mmol of
1M Li tert-BuO. The temperature was increased to room temperature
again and most of THF was evaporated using Rotovap. The reaction
mixture was added with 50 mL of water and extracted using 100 mL of
MTBE three times. The combined MTBE phase was dried over
Na.sub.2SO.sub.4, and the solvent was removed using Rotovap to
obtain the crude TIPS-1,3-diphenyl-3-TIPS-1-dipropanediol. The
crude TIPS capped diol was dissolved in 50 mL of THF. After bubbled
THF solution with N.sub.2, 26 mL 1M TBAF in THF was added. The
reaction was run at room temperature for 1 h in a N.sub.2
atmosphere, and then 4 mL of Acetic acid was added. The THF was
removed using Rotovap and the residue was dissolved in 100 mL of
MTBE and washed using 30 mL of aqueous NH.sub.4Cl solution three
times. The product was extracted from the MTBE phase using 30 mL
10% NaOH three times. The product was immediately released using
concentrated HCl and extracted using MTBE (80 mL, five times). The
MTBE phase was washed with 50 mL of water three times and 30 mL of
brine three times, and then dried over Na.sub.2SO.sub.4. MTBE was
removed using Rotovap, 4 g of product (18-xiv) was obtained after
being dried in a vacuum. .sup.1H NMR (400 MHz. CHLOROFORM-d)
.delta.7.29-7.06 (m, 10H), 4.42 (dd, J=9.1, 4.3 Hz, 1H), 4.11 (dt,
J=9.1, 6.1 Hz, 1H), 2.38-2.16 (m, 2H).
Procedure 3:
##STR00494##
[0809] Diol protection using thiocarbonyl group and rearrangement
of the thiocarbonyl protective group: After 10 mmol (2.28 g) of
1,3-diphenyl-dipropanediol (18-xii) and 11 mmol of
thiocarbonyldiimidzaole dissolved in 50 mL of THF, the temperature
was decreased to -78.degree. C. Subsequently, 20 mmol of 1M Li
tert-BuO/THF was added, followed by removing reaction to room
temperature. The reaction mixture was diluted using 100 mL of MTBE
and quenched using 50 mL of citric acid (10%). The organic layer
was washed with NaHCO.sub.3 (5%), water and brine before being
dried over Na.sub.2SO.sub.4. After filtering the solid out, the
organic solution was added with 1 equiv. of tetrabutylammonium
iodide. The solution was refluxed for 2 h. The solvent was removed
using Rotovap and 2 g of purified rearrangement product was
obtained via a silica column (EA:Hexane=1:4).
Synthesis of (S,S)-1,3-diphenyl-3-hydroxyl-1-propanethiol
[0810] The 2 g of purified rearrangement product was dissolved in
50 mL of THF. And then 4 equiv. LiAiH.sub.4 was injected at
0.degree. C. The solution was stirred for 1 h. The reaction was
quenched using 10 mL of EA followed by 10 mL of water at 0.degree.
C. After the mixture was poured into a beaker containing 15 mL
concentrated HCl and 100 g of ice, 100 mL of MTBE was immediately
added. Subsequently, the aqueous layer was separated and extracted
using 40 mL of MTBE for another three times. The combined organic
layer was washed using 50 mL brine three times. After being dried
over Na.sub.2SO.sub.4, the solid was removed via filtration and
solvent was removed using Rotovap. At last, 1.2 g of
(S,S)-1,3-diphenyl-3-hydroxyl-1-propanethiol (18-xvi) was obtained
using Silica column (EA:Hexane=1:4).
c. Step 4
##STR00495##
[0812] POCl.sub.3 (0.54 mL, 1.1 equiv.) in THF (35 mL) was cooled
to -78.degree. C. A solution of mercaptopropanol (18-vi, 1.36 g,
1.0 equiv.) and Et.sub.3N (1.68 mL, 2.2 equiv.) in THF (20 mL) was
prepared and added dropwise to the POCl.sub.3 solution at
-78.degree. C. After stirring for 1 hour at this temperate, an
aliquot was analyzed by .sup.31P NNR. The solution was then warmed
to room temperature and Et.sub.3N (7.7 mL, 10 equiv.) and water
(1.0 mL, 10 equiv.) were added. After stirring for 10 minutes the
reaction was partitioned between water and EtOAc. The organic layer
was washed with water (3.times.) and finally brine. The aqueous
layer (after saturating with brine) was back extracted multiple
times with DCM (.about.600 mL for this scale) and the organics were
pooled, dried and concentrated giving pure triethylammonium salt
(1.36 g, 60% yield) as a sticky goo.
Compounds 1-45 or 1-46
##STR00496##
[0814] The title compounds were made by using the same chemistry as
the preparation of compound 1-44. .sup.1H NMR (400 MHz,
Chloroform-d) .delta. 7.57-7.50 (m, 1H), 7.41-7.13 (m, 10H), 5.56
(dq, J=7.7, 3.7 Hz, 1H), 4.65 (dt, J=11.4, 2.7 Hz, 0.5H), 4.53 (dt.
J=12.6, 6.5 Hz, 0.5H), 2.98 (dq, J=10.9, 6.9, 4.9 Hz, 6H), 2.40
(tdd, J=14.3, 8.5, 5.4 Hz, 1H), 2.30-2.15 (m, 1H), 1.25 (t, J=7.3
Hz, 9H). .sup.31P NMR (162 MHz, Chloroform-d) .delta. 13.80,
11.33.
Compounds 1-47 or 1-48
##STR00497##
[0816] .sup.1H NMR (400 MHz, Chloroform-d) .delta. 7.61-7.11 (m,
10H), 5.55 (td, J=8.9, 4.5 Hz, 1H), 4.57 (ddd, J=11.3, 8.4, 5.6 Hz,
1H), 2.61-2.14 (m, 2H). .sup.31P NMR (162 MHz, Methanol-d.sub.4)
.delta. 16.03, 12.56.
Compound 1-49
##STR00498##
[0818] To a cooled solution of POC.sub.3 (1.1 equiv.) in THF (0.1
M) at -78.degree. C. was added dropwise a solution of
mercaptopropanol (18-6, 1.0 equiv.) and NEt.sub.3 (2.2 equiv.) in
THF (0.1 M) over 5 min. After stirring for 1 hr, the reaction was
warmed to rt and an aliquot was checked by .sup.31P NMR.>90%
product detected. Filtration of the precipitated salts followed by
concentration afforded the product.
Compound 1-50
##STR00499##
[0820] The title compounds were made by using the same chemistry as
preparation of compound 1-49. .sup.1H NMR (400 MHz, CHLOROFORM-d)
7.48-7.28 (m, 11H), 5.89 (br dd, J=11.6, 2.3 Hz, 1H), 5.08-4.96 (m,
1H), 2.81 (dt, J=15.1, 12.1 Hz, 1H), 2.53 (br d, J=15.1 Hz, 1H).
.sup.31P NMR (202 MHz, CHLOROFORM-d) 26.0 (s, 1P).
Compound 1-51
##STR00500##
[0822] The title compounds were made by using the same chemistry as
preparation of compound 18-7f. .sup.1H NMR (400 MHz, CHLOROFORM-d)
.delta.7.52-7.35 (m, 10H), 5.77 (td, J=8.4, 3.6 Hz, 1H), 4.93-4.86
(m, 1H), 2.97 (ddd, J=15.2, 7.2, 3.7 Hz, 1H), 2.78-2.71 (m,
1H).
General Procedures to Make Compounds 1-52 and 1-53
##STR00501##
[0824] To a cooled solution of P(V)Cl (1.5 equiv.) and HXAr (1.0
equiv.) in MeCN (0.1 M) at 0.degree. C. was added dropwise DBU (1.2
equiv.) After stirring for 1 hr, the reaction was warmed to rt. The
precipitated salts were filtered and the product isolated after
column chromatography.
Compound 1-52
##STR00502##
[0826] .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.8.37-8.29 (m,
J=9.0 Hz, 2H), 7.60-7.54 (m, J=8.9 Hz, 2H), 7.50-7.33 (m, 10H),
5.75 (br d, J=11.4 Hz, 1H), 4.67 (br d, J=12.1 Hz, 1H), 2.68-2.58
(m, 1H), 2.56-2.45 (min, 1H). .sup.31P NMR (202 MHz. CHLOROFORM-d)
.delta.12.1 (s, 1P).
Compound 1-53
##STR00503##
[0828] .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.8.34 (d, J=9.0
Hz, 1H), 8.19-8.15 (m, J=9.0 Hz, 2H), 7.49-7.37 (m, 11H), 7.11-7.07
(m, J=9.0 Hz, 2H), 5.95-5.90 (m, 1H), 4.86-4.79 (m, 1H), 2.95 (br
dd, J=15.6, 5.3 Hz, 1H), 2.76 (ddd, J=15.3, 10.3, 4.5 Hz, 1H).
.sup.31P NMR (202 MHz, CHLOROFORM-d) .delta.12.5 (s, 1P).
d. Step 5
[0829] General Procedure A to Prepare Loaded Nucleosides
##STR00504##
[0830] To a solution of P(v) reagent (excess) and nucleoside (1.0
equiv.) in MeCN or THF or Pyridine or DMF (0.1 M) was added
Condensation Reagent, Base and Activator (excess.) The reaction
proceeds sometimes with heat or at room temperature for 1-48 hours.
The product is isolated after column chromatography.
[0831] Specific conditions that work the best: Triisopropylbenzene
sulfonyl chloride or mesityl sulfonyl chloride as condensation
reagents with N-Methyl Imidazole as base in THF at 50/60.degree.
C.
Compound 3-132
##STR00505##
[0833] .sup.1H NMR (600 MHz, Acetone-d.sub.6) .delta. 10.07 (s,
1H), 7.64-7.56 (m, 2H), 7.56-7.47 (m, 5H), 7.50-7.34 (m, 11H),
7.37-7.28 (m, 2H), 7.28-7.21 (m, 1H), 6.96-6.84 (m, 4H), 6.42 (dt,
J=8.3, 6.3 Hz, 1H), 5.91-5.82 (m, 1H), 5.43 (dddt, J=31.4, 8.6,
5.5, 2.6 Hz, 1H), 4.85 (q, J=7.7 Hz, 0H), 4.76 (q, J=7.6 Hz, 1H),
4.39-4.34 (m, 1H), 3.83-3.74 (m, 7H), 3.55-3.44 (m, 2H), 2.78-2.60
(m, 4H), 1.52 (dd, J=18.6, 1.2 Hz, 3H), 1.36-1.23 (m, 3H). .sup.31P
NMR (162 MHz, Acetone-d6) .delta. 21.08 (d, J=10.0 Hz).
General Procedure B to Prepare Loaded Nucleosides
##STR00506##
[0835] To a solution of P(V) reagent (1.3 equiv.) and nucleoside
(1.0 equiv.) in MeCN (0.1 M) was added dropwise DBU (1.3 equiv.)
After stirring for 0.5 hr. The product was worked up as usual and
the product isolated after column chromatography.
Compound 3-133
##STR00507##
[0837] .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.8.32 (s, 1H),
7.57 (br t, J=8.4 Hz, 4H), 7.38-7.23 (m, 16H), 6.42 (dd, J=9.1, 5.1
Hz, 1H), 5.67 (br d, J=11.3 Hz, 1H), 5.40 (br t, J=6.5 Hz, 1H),
4.83 (br d, J=11.9 Hz, 1H), 4.22 (br s, 1H), 3.93 (br d, J=11.6 Hz,
1H), 3.85 (br d, J=11.4 Hz, 1H), 2.59 (br dd, J=13.8, 5.0 Hz, 1H),
2.50-2.38 (m, 1H), 2.38-2.30 (m, 1H), 2.30-2.19 (m, 1H), 1.02 (s,
9H). .sup.31P NMR (202 MHz, CHLOROFORM-d) .delta. 20.5 (s, 1P)
Compound 3-134
##STR00508##
[0839] .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.7.66 (br d, J=7.0
Hz, 4H), 7.48-7.34 (m, 16H), 6.58 (dd, J=9.2, 5.0 Hz, 1H), 5.57 (br
d, J=11.4 Hz, 1H), 5.41 (br t, J=6.7 Hz, 1H), 4.54 (br d, J=11.6
Hz, 1H), 4.45 (s, 1H), 4.08-3.99 (m, 2H), 2.85 (dd, J=13.8, 5.0 Hz,
1H), 2.64-2.46 (m, 1H), 2.45-2.29 (m, 2H), 1.16-1.06 (m, 9H).
.sup.31P NMR (202 MHz, CHLOROFORM-d) .delta.15.8 (s, 1P).
Compound 3-135
##STR00509##
[0841] .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.8.09 (br s, 1H),
7.70-7.62 (m, 4H), 7.54 (br d, J=7.6 Hz, 1H), 7.50-7.35 (m, 14H),
7.27-7.22 (m, 1H), 6.38-6.28 (m, 1H), 5.79 (br s, 1H), 5.18 (br d,
J=6.1 Hz, 1H), 4.76 (br d, J=19.8 Hz, 1H), 3.95 (s, 1H), 3.72-3.67
(m, 1H), 2.88-2.76 (m, 1H), 2.67 (br dd, J=9.8, 5.2 Hz, 1H), 1.12
(s, 5H), 1.09 (s, 4H). .sup.31P NMR (202 MHz, CHLOROFORM-d)
.delta.17.9 (s, 1P), 17.6 (s, 1P).
e. Step 6
##STR00510##
[0843] To a solution of P(V) reagent (1.0 equiv.) and nucleoside
(2.0 equiv.) in MeCN (0.1 M) was added dropwise DBU (3.0 equiv.)
After stirring for 1 hr. The product was worked up as usual and the
product isolated after column chromatography.
Compound 5-11
##STR00511##
[0845] .sup.31P NMR (202 MHz, CHLOROFORM-d) .delta. -1.74 (s,
1P).
Compound 5-12
##STR00512##
[0847] .sup.31P NMR (202 MHz, MeCN-d) .delta. -0.81 (s, 1P).
[0848] It is to be appreciated that the Detailed Description
section, and not the Summary and Abstract sections, is intended to
be used to interpret the claims. The Summary and Abstract sections
may set forth one or more but not all exemplary embodiments of the
present disclosure as contemplated by the inventor(s), and thus,
are not intended to limit the present invention and the appended
claims in any way.
[0849] The present invention has been described above with the aid
of functional building blocks illustrating the implementation of
specified functions and relationships thereof. The boundaries of
these functional building blocks have been arbitrarily defined
herein for the convenience of the description. Alternate boundaries
can be defined so long as the specified functions and relationships
thereof are appropriately performed.
[0850] The foregoing description of the specific embodiments will
so fully reveal the general nature of the invention that others
can, by applying knowledge within the skill of the art, readily
modify and/or adapt for various applications such specific
embodiments, without undue experimentation, without departing from
the general concept of the present disclosure. Therefore, such
adaptations and modifications are intended to be within the meaning
and range of equivalents of the disclosed embodiments, based on the
teaching and guidance presented herein. It is to be understood that
the phraseology or terminology herein is for the purpose of
description and not of limitation, such that the terminology or
phraseology of the present specification is to be interpreted by
the skilled artisan in light of the teachings and guidance.
[0851] The breadth and scope of the present disclosure should not
be limited by any of the above-described exemplary embodiments, but
should be defined only in accordance with the following claims and
their equivalents.
* * * * *